Thermospray Mass Spectrometry Research Articles

A Rare Mass Spectrometric Neutral Loss of 48 u

Fast atom bombardment and thermospray mass spectrometry results are consistent with the 2-deoxyfluoromethine derivative of the nucleoside (cytidine) shown below and designated 2DFC. However, a mass loss of 48 u from [M+H]+ (m/z 258) was observed forming an abundant fragment ion at m/z 210. The loss seemed peculiar since a 48 u neutral is more often associated with H3CSH or H3SiOH, which are not possible neutrals to be eliminated from 2DFC. Accurate mass measurement of m/z 210 indicated the 48 u neutral lost had the molecular formula CH4O2. Constant neutral loss (48 u) linked scan experiments showed the fragmentation (depicted below) is concerted within the microsecond time frame of the mass spectrometer. Other structurally homologous nucleosides, such as cytidine, do not exhibit this prominent fragmentation. Computerized molecular modeling was used to propose why the marked elimination occurs with 2DFC. © 1997 by John Wiley & Sons, Ltd.

Determination of furazolidone in animal feeds using liquid chromatography with UV and thermospray mass spectrometric detection

A method is presented for the detection of the nitrofuran antibacterial, furazolidone in animal feeds. Following solvent extraction of feed samples, the extracts were purified on an alumina column. The column eluents were separated by HPLC using a reversed phase column and detected either by UV monitoring at 365 nm, or by thermospray mass spectrometry, monitoring the ion at m/z 243. The LC-UV procedure was used to quantify furazolidone at medicated levels (200 mg/kg) and at contamination levels of 5 and 20 mg/kg. The limit of detection was 1 mg/kg. Overall mean recoveries from fortified samples were, 93.4, 98.2 and 98.0% at concentrations of 5, 20 and 200 mg/kg, respectively. The mass spectrometric procedure was used to analyse sample extracts at concentrations <5mg/kg. The overall recovery from feed samples fortified at 1 mg/kg was 93.8%. The assay is therefore capable of the determination of furazolidone in animal feeds at both high medicated concentrations and at low contamination levels.

Detection of Corticosteroids in the Presence of Organic Acids in a Liquid Chromatography Eluent Using the Negative-ion Mode of Thermospray Mass Spectrometry

The pKa values of organic acids (HCOOH, CH3CH2COOH, HOCH2COOH, HSCH2COOH and FCH2COOH) in a liquid chromatography (LC) eluent mainly influenced the generation of adduct ions with corticosteroids. The relative abundance of [M + R] adduct ions increased as the pKa value of the organic acid decreased. An interaction between the functional groups of the organic acids in the LC eluent and those of the corticosteroid affected the generation of fragment ions. When HCOOH was present in the mobile phase, the sensitivity to endogenous hydrocortisone and cortisone in human urine was maximized.

Liquid Chromatography/Thermospray/Mass Spectrometry Analysis of Beauvericin

Beauvericin (BEA) is a bioactive hexadepsipeptide coproduced by the same Fusarium species that produce fumonisin mycotoxins. BEA is insecticidal and antimicrobial, and, being lipophilic, has the potential to bioaccumulate. Its mammalian toxicity has only recently been characterized, and sensitive analytical methodology to analyze for BEA is lacking. On-line thermospray mass spectrometry (LC/TSP/MS) was used to analyze BEA. A reversed-phase column and a mobile phase that consisted of CH3CN/0.4 M HCO2NH4 were used to resolve BEA. Positive ion spectra yielded signals at m/z 785 (M + H)+, m/z 802 (M + NH4)+, and m/z 823 (M + K)+. Standard curves plotted in the range 10−100 ng for the ions m/z 785, 802, and 823 had coefficients of determination (r2) of 0.996, 0.992, and 0.999. The lowest detectable limit was 1 ng at a S/N of 5:1. The developed LC/TSP/MS method was used successfully to analyze Fusarium proliferatum-fermented cracked corn containing BEA and spiked meat samples. Keywords: Beauvericin; fumonisins;...

Quantification of corticosteroids in human plasma by liquid chromatography–thermospray mass spectrometry using stable isotope dilution

Liquid chromatography–thermospray mass spectrometry (LC–TSP-MS) using isotope dilution was investigated for quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma. Complete separation attained by a LiChroCART Supersupher reversed-phase column and elution with 0.05 M ammonium formate–tetrahydrofuran–methanol (180:53:17, v/v/v) resulted in a significantly large isotope effect of the deuterium-labeled analogs on the HPLC behavior and caused difficulty in quantification. Reduction of the isotope effect on the retention times using 0.05 M ammonium formate–acetonitrile (65:35, v/v) permitted accurate quantification of cortisol and cortisone by the isotope dilution LC–TSP-MS, although separation between cortisol and prednisone was incomplete.

Metabolism of 4-[1-(2-fluoro-4-biphenylyl)ethyl]-2-methylaminothiazole (SM-8849) in rats.

Metabolism of 4-[1-(2-fluoro-4-biphenylyl)ethyl]-2-methylaminothiazole (SM-8849), a novel immunomodulatory agent, in rats was investigated. By co-chromatography with authentic samples, desmethylated (SM-8800) p-hydroxylated (SL-5512) and desmethylated-p-hydroxylated SM-8849 (SL-5515) were detected in the bile. Thermospray mass spectrometry (TSP-MS) analysis of five metabolites isolated from the bile revealed molecular ions of both conjugates (glucuronides and a sulfate) and their aglycones. Aglycone structures were determined by comparison of their product spectra with those of authentic standards. Further analyses of conjugation sites were carried out by 1H-NMR including differential NOE. As a result, the sulfate of SL-5515 (5515-S), the N-glucuronides of SL-5512 and SM-8849 (5512-NG and 8849-NG, respectively), the glucuronide of SL-5512 (5512-G) and the O-glucuronide of 4-hydroxy-3-methoxy-SM-8849 (CatOMe-OG) were identified. In addition, N-methylthiouras was identified in urine by LC/MS/MS.

Determination of dimetridazole in poultry tissues and eggs using liquid chromatography-thermospray mass spectrometry.

A method is presented for the determination of the nitroimidazole drug dimetridazole (DMZ) in poultry tissues and eggs by liquid chromatography (LC)-thermospray mass spectrometry (MS). Deuteriated DMZ was employed as an internal standard. Samples were extracted with dichloromethane (muscle) or toluene (liver, egg) and applied to silica gel cartridges. Dimetridazole was eluted with acetone and the eluate evaporated to dryness at 40 degrees C under nitrogen. The residue was redissolved in methanol-water (1 + 1, v/v) and washed with hexane before LC-MS analysis. Quantification was by the ratios of the positive [M + H]+ ions at m/z 142 and 145 for DMZ and the internal standard, respectively. Internal standard corrected recoveries were between 93 and 102% with RSDs between 1.2 and 7.7% for liver spiked at 5, 10 and 20 ng g-1 and muscle and eggs spiked at 5 ng g-1. Absolute recoveries were approximately 80%. The method is suitable for statutory residue testing and was used to measure DMZ residues in eggs from chickens fed a diet containing DMZ at 10 mg kg-1.

Determination of trimethoprim in tissues using liquid chromatography-thermospray mass spectrometry.

A method is described for the determination of the antibacterial drug trimethoprim in tissues. Minced tissue is homogenised with chloroform-acetone (1 + 1 v/v), filtered, and the filtrate evaporated to an oily residue using a rotary evaporator. The residue is redissolved in methanol-water-acetic acid (50 + 48.7 + 1.3 v/v) and any fats present are partitioned into hexane. The aqueous phase is analysed by liquid chromatography-thermospray mass spectrometry in positive mode with the protonated molecular ion at m/z 291 being monitored. Recoveries ranged between 60% in liver and 79% in muscle. The limit of determination was 25 micrograms kg-1 and the limit of detection was approximately 4 micrograms kg-1. The method is suitable for monitoring tissues taken under national surveillance schemes for veterinary drug residues.

On-line solid-phase extraction in liquid chromatography using restricted access pre-columns for the analysis of s-triazines in humic-containing waters

A coupled-column liquid chromatographic system using solid-phase extraction pre-columns for the seven chlorotriazines, atrazine, desethylatrazine, desisopropylatrzine (DIA), propazine, simaize, desthylterbythylazine and terbuthylazine, using photodiode-array detection (PDAD) was developed. Restricted access (RA), was investigated as a pre-column material, making use of non-adsorptive size exclusion of macromolecules and simultaneous dynamic portioning of the analytes, for the pre-concentration in environmental analysis. The RA phase was characterized and shown to solve problems with difficult matrix effects in humic-containing surface water samples. Other pre-column materials, e.g. reversed-phase C 18, mono-functional C 18, extraction disk mixture of C 18 and styrene-divinylbenzene (PS-DVB) and LiChrolut EN, a polymeric sorbent specially designed for environmental analysis, were investigated and compared for their selectivity and stability, respectively. Comparisons were made with respect to selectivity and efficiency for the SPE columns separately and in combination with analytical columns. Recovery data and matrix dependencies were studied at concentration levels ranging between 0.1–10 μg/1. The RA pre-columns showed superior stability in complex environmental matrices, such as reference humic water and river water, due to the restriction of macromolecules. Retention properties with the RA alkyl-diol-silica were not sufficient for the most polar metabolites. However, efficient sorption/desorption kinetics were found with medium-polar and non-polar compounds. LiChrolut EN was found to strongly retain these polar metabolites as well as medium and non-polar solutes, and was selected for a deeper investigation (e.g. salinity dependence and pH). For a 10×2 mm I.D. pre-column, the breakthrough volume for the polar metabolite DIA was at least 150 ml, while for the RA pre-columns it was less than 10 ml. Unfortunately, this polymeric support was strongly effected, in terms of stability, in complex matrices which lead to repeated and expensive exchanging of pre-columns. However, in order to detect even the polar metabolites these pre-columns were finally used on-line together with liquid chromatography PDAD and thermospray mass spectrometry for the determination of triazines in water samples from the Ebro river during different seasons of the year.

Adduct Ion Formation by Aromatic Amines in Thermospray Mass Spectrometry

The formation of solvent adduct ions in thermospray and ionspray mass spectrometry was studied for twelve aromatic amines : aniline, N-methylaniline, N,N-dimethylaniline, 3-aminophenol, 3-methylaminophenol, 3-dimethylaminophenol, 2-aminopyridine, 2-methylaminopyridine and 2-dimethylaminopyridine, 2-amino-5,6-dimethyl-4-hydroxypyrimidine, 2-methylamino-5,6-dimethyl-4- hydroxypyrimidine and 2-dimethyl-amino-5,6-dimethyl-4-hydroxypyrimidine. For all compounds, adduct ions, [M + H + A n ] + , with A being methanol or acetonitrile, were observed in the thermospray mass spectra ; water adduct ions were observed for a few compounds. No adduct ions with ammonia were formed when ammonium acetate was added to the liquid chromatographic carrier stream. These observations cannot be explained on the basis of gas-phase ion-molecule reactions of the neutral analyte and protonated solvent or solvent additive molecules. Comparative experiments, changing the pH of the carrier stream in both thermospray and ionspray ionization, showed that the solvent adduct ions present in the thermospray mass spectra are not likely to be formed by ion evaporation processes. Incomplete evaporation of droplets or cluster ions is proposed to be responsible for the observations. With this hypothesis, both the absence of ammonium adduct ions and the dependence of the adduct ion abundances on the N-methylation can be related to the adduct-analyte bond strengths.

Determination of ten carbamate pesticides in aquatic and sediment samples by liquid chromatography-ionspray and thermospray mass spectrometry

Ten carbamate pesticides which exhibit large differences in polarity were determined simultaneously in various environmental samples, using both column liquid chromatography (LC)-thermospray (TSP) mass spectrometry (MS) and LC-ionspray (ISP) MS. For sample clean-up, column chromatography with three stationary phases, neutral aluminium oxide, Florisil and aminopropyl-bonded modified silica, were tested. The aminopropyl stationary phase showed the best results, with acetone-dichloromethane (25:75) as eluent; analyte recoveries were 76–100% for all compounds with a relative standard deviation of 2–8%. In the ISP mass spectra of eight of the ten carbamates, the sodium adduct ion, [M + Na] +, was the base peak, while the protonated molecule, [M + H] +, was the most abundant ion with carbendazim and aminocarb. An eluent flow-rate between 100 and 300 μl/min was found to be optimum, with optimized nebulizer and drying gas flow-rates of 350 and 15 l/h, respectively. A capillary voltage of 3.5 kV resulted in the largest total ion current. The optimum extraction voltage with regard to analyte detectability and confirmation purposes was between 15 and 35 V. The detection limits obtained with the LC-ISP-MS system were typically in the 10–60 pg range (10 μl of 10 μg/l standard solutions), which is 10–150-fold better than obtained with LC-TSP-MS (selected-ion monitoring mode used in both instances). Large-volume injections of tap water spiked with selected carbamates at a level of 0.1 μg/l illustrated the potential of LC-ISP-MS with respect to analyte detectability. Furthermore, carbofuran was identified at a concentration level of ca. 5 μg/l in water samples from the Ebro delta. LC-TSP-MS of oxamyl and methomyl in a sediment sample containing a high percentage of organic matter was adversely affected by the presence of co-extractives. This problem did not occur with LC-ISP-MS.

Adduct Ion Formation by Aromatic Amines in Thermospray Mass Spectrometry

The formation of solvent adduct ions in thermospray and ionspray mass spectrometry was studied for twelve aromatic amines: aniline,N-methylaniline,N,N-dimethylaniline, 3-aminophenol, 3-methylaminophenol, 3-dimethylaminophenol, 2-aminopyridine, 2-methylaminopyridine and 2-dimethylaminopyridine, 2-amino-5,6-dimethyl-4-hydroxypyrimidine, 2-methylamino-5,6-dimethyl-4- hydroxypyrimidine and 2-dimethyl-amino-5,6-dimethyl-4-hydroxypyrimidine. For all compounds, adduct ions, [M+H+An]+, with A being methanol or acetonitrile, were observed in the thermospray mass spectra; water adduct ions were observed for a few compounds. No adduct ions with ammonia were formed when ammonium acetate was added to the liquid chromatographic carrier stream. These observations cannot be explained on the basis of gas-phase ion–molecule reactions of the neutral analyte and protonated solvent or solvent additive molecules. Comparative experiments, changing the pH of the carrier stream in both thermospray and ionspray ionization, showed that the solvent adduct ions present in the thermospray mass spectra are not likely to be formed by ion evaporation processes. Incomplete evaporation of droplets or cluster ions is proposed to be responsible for the observations. With this hypothesis, both the absence of ammonium adduct ions and the dependence of the adduct ion abundances on the N-methylation can be related to the adduct–analyte bond strengths.

Determination of the dihydropyridine aryloxypropanolamine compound Z6568 and four metabolites in plasma by liquid chromatography/negative-ion mass spectrometry.

A sensitive and selective liquid chromatographic/mass spectrometric assay for the determination of the dihydropyridine aryloxypropanolamine compound Z6568 and four metabolites in plasma was developed and validated. Z6568 and the metabolites were isolated from plasma by a simple solvent extraction procedure and separated by reversed-phase high-speed liquid chromatography combined with thermospray mass spectrometry. Filament ionization was used and the molecular negative ions were monitored. The analysis of spiked plasma demonstrated the good accuracy and precision of the assay with a detection limit of 0.5 ng ml-1 for Z6568. Data relative to the optimization of the mass spectrometric conditions and the validation protocol are reported. the method has been used for pharmacokinetic studies in laboratory animals.

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.