Structural determination of cyclitol derivatives by fast-atom bombardment tandem mass spectrometry

Three cyclitol derivatives were isolated from the marine sponge Sarcotragus sp. by reversed-phase high-performance liquid chromatography and analyzed by fast-atom bombardment mass spectrometry (FAB-MS). Their structural elucidation was carried out with FAB tandem mass spectrometry (FAB-MS/MS). FAB-MS spectra produced a significant abundance of the sodium adducts [M+Na]+ and [M+2Na-H]+ from a mixture of m-NBA and NaI. In addition, trifluoroacetylation of the cyclitol derivatives was used for confirmation of the presence of the cyclitol ring. High abundance [M-5H+5CF3CO+Na]+ ions were observed in the FAB-MS spectra of the trifluoroacetyl-cyclitol derivatives. Collision-induced dissociation (CID) of the [M+Na]+ ions produced diverse product ions via a series of dissociative processes. Charge-remote fragmentation (CRF) patterns of [M+Na]+ ions were very useful for the identification of product ions which are characteristic for the cyclitol ring and long hydrocarbon chains substituted at the glycerol backbone. Moreover, the CID-MS/MS spectra of the [M+Na]+ ions yielded characteristic product ions at m/z 53, 83, 113, 155 and 171 for the cyclitol moiety, and at m/z 213, 229 and 245 for the glycerol backbone attached to the cyclitol ring.     

Structural determination of sphingomyelin by tandem mass spectrometry with electrospray ionization

Alkaline metal adduct ions of sphingomyelin were formed by electrospray ionization in positive ion mode. Under low energy collisionally activated dissociation (CAD), the product ion spectra yield abundant fragment ions representative of both long chain base and fatty acid which permit unequivocal determination of the structure. Tandem spectra obtained by constant neutral loss scanning permit identification of sphingomyelin class and specific long chain base subclass in the mixture. The fragmentation pathways under CAD were proposed, and were further confirmed by source CAD tandem mass spectrometry. The total analysis of sphingomyelin mixtures from bovine brain, bovine erythrocytes, and chicken egg yolk is also presented.     

Comparison of flow injection analysis electrospray mass spectrometry and tandem mass spectrometry and electrospray high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry for the determination of underivatized amino acids

Twenty proteinogenic amino acids (AAs) were determined without derivatization using flow injection analysis followed by electrospray ionization mass spectrometry and tandem mass spectrometry (ESI-MS and ESI-MS/MS) and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry (ESI-FAIMS-MS and ESI-FAIMS-MS/MS), in positive and negative ionization modes. Three separate sets of ESI-FAIMS conditions were used for the separation and detection of the 20 AAs. Typically ESI-FAIMS-MS showed somewhat improved sensitivity and significantly better signal-to-noise ratios than ESI-MS mainly due to the elimination of background noise. However, the difference between ESI-FAIMS-MS and ESI-MS/MS was significantly less. ESI-FAIMS was able to partially or completely resolve all the isobaric amino acid overlaps such as leucine, isoleucine and hydroxyproline or lysine and glutamine. Detection limits for the amino acids in ESI-FAIMS-MS mode ranged from 2 ng/mL for proline to 200 ng/mL for aspartic acid. Overall, ESI-FAIMS-MS is the preferred method for the quantitative analysis of AAs in a hydrolyzed yeast matrix.     

Quantitative determination of ochratoxin A by liquid chromatography/electrospray tandem mass spectrometry

Mass spectrometry of ochratoxin A (OTA) and B (OTB) under electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) was studied. ESI offers higher sensitivities and less fragmentation than APCI. A sensitive LC/MS/MS method for the determination of ochratoxin A (OTA) in human plasma samples was developed. The absolute minimum detection limit was around 10-20 pg per injection, corresponding to 0.5 ppb in an injection equivalent to 20-40microg of human plasma. Ochratoxin B (OTB) was used as an internal standard and its absence in real-life samples was carefully checked before samples were spiked with the internal standard. It was found that these two ochratoxins are susceptible to sodium adduct formation. Fragment ions from the [M + H](+) and [M + Na](+) ions of both OTA and OTB were monitored in the multiple reaction monitoring mode. Three quantitative approaches, standard addition method, internal standard method (using ochratoxin B as an internal standard) and external standard method, were compared in the analysis of human blood plasma. Results from the mass spectrometric method were comparable to those from a conventional LC/fluorescence method. The LC/MS/MS method was also applied to the analysis of contaminated coffee samples.     

Structural determination of hexadecanoic lysophosphatidylcholine regioisomers by fast atom bombardment tandem mass spectrometry

The structural determination of sn-1 and sn-2 hexadecanoic lysophosphatidylcholine (LPC) regioisomers was carried out using fast atom bombardment tandem mass spectrometry (FAB-MS/MS). The collision-induced dissociation (CID) of protonated and sodiated molecules produced diverse product ions due mainly to charge remote fragmentations. Based on the information obtained from the CID spectra of protonated and sodiated molecules, sn-1 and sn-2 hexadecanoic LPC isomers could be discriminated. Especially, the abundance ratio of the diagnostic ion pair [m/z 224/226] in the CID spectra of [M + H](+) ions was shown to be greatly different. Moreover, the CID-MS/MS spectra of sodium-adducted molecules for hexadecanoic LPC isomers showed characteristic product ions such as [M + Na - 103](+), [M + Na - 85](+), and [M + Na - 59](+), by which their regio-specificity can be differentiated.     

Sequencing membrane proteins by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) is an attractive technique for sequencing membrane proteins because it can be applied to peptides in mixtures that are difficult to separate chromatographically. To evaluate the suitability of MS/MS sequencing for membrane proteins and to develop protocols for the preparation of the cleaved peptides, we employed the well characterized apoproteins of bacteriorhodopsin and bovine rhodopsin, i.e. bacterioopsin and opsin, respectively. Without separation, nine out of ten peptides resulting from cyanogen bromide cleavage of bacterioopsin were detected by fast atom bombardment MS, the single undetected fragment being a tetrapeptide that was presumably hidden in the low-m/z matrix background. Furthermore, MS/MS was used to confirm the sequence of all the peptides detected with m/z values below 3.5 kDa (40% of the protein). Bovine opsin was analyzed in a similar fashion. Tandem MS/MS has thus allowed the sequencing of substantial portions of two integral membrane proteins by the analysis of unseparated peptide mixtures, demonstrating for the first time that this technique can obviate some of the most serious difficulties associated with sequencing membrane proteins, namely the difficult-to-achieve separation of the ‘sticky’ peptide fragments.     

Structure elucidation of phase II metabolites by tandem mass spectrometry: an overview

The present paper provides a summary of the collision-induced dissociation of protonated and deprotonated phase II metabolites of drugs and pesticides. This overview is based on published literature and unpublished data from the authors. In particular, glutathione conjugates and their biotransformation products are discussed in detail. In addition, the fragmentation of the major classes of conjugates, i.e. glucuronides, glucosides, malonylglucosides, sulfates, acetates, methyl and glycine conjugates, is reported. Collision-induced dissociation, as studied by tandem mass spectrometry, allows the rapid identification of the type of conjugate, whereas the exact conjugation site can in general be determined only by additional NMR experiments.     

AutoScan: an automated workstation for rapid determination of mass and tandem mass spectrometry conditions for quantitative bioanalytical mass spectrometry

An automated flow injection analysis (FIA) mass spectrometry system (AutoScan) was developed to allow rapid unattended determination of optimal conditions during mass (ms) and tandem mass spectrometry (ms/ms) on new chemical entities (NCEs) arranged in 96-well plates. The 96-well plate is placed on the deck of a modified Gilson Multiprobe autosampler for injection into a PE Sciex API 2000 triple quadrupole mass spectrometer. A customized software interface is used to create the necessary scan experiments by associating each 96-well plate of NCEs to be scanned with an index file containing data on the identity of each analyte and its expected molecular weight. Analytes are injected four at a time into a custom injection manifold and conventional mass spectra are acquired in both polarities (+/-) using an alternating positive/negative Q1 scan function. The software determines the optimal polarity and definitive precursor ion for all analytes and uses the results to build the injection sequence for product ion scanning. The samples are automatically re-injected under MS/MS conditions, and product ion scans that loop among different collision energies are collected for each analyte. The resulting data are processed automatically and the optimal MS/MS transitions for each analyte are selected. A color-coded graphical interface facilitates data review. Any unusual ion transitions or transposition errors made during plate preparation are noted and corrected. Complete MS and MS/MS conditions are obtained for 96 compounds in about one hour and the resulting data are available for download as sample control injection sequence files.     

Multiresidue determination of pesticides in fruit and vegetables by gas chromatography/tandem mass spectrometry

Pesticide residues in fruit and vegetables were determined by gas chromatography/tandem mass spectrometry (GC/MS/MS). Electron impact (EI)/MS/MS and chemical ionization (CI)/MS/MS were developed for 80 compounds, including organochlorine, organophosphorus, organonitrogen, and pyrethroids, providing unambiguous spectral confirmation for these complex matrixes. Residues were extracted from samples with acetone followed by a mixture of dichloromethane-petroleum ether. Two injections per sample were required for analysis of the entire pesticide list by EI/MS/MS and CI/MS/MS. Initial steps involving cleanup and concentration of extracts were eliminated. The excellent selectivity and good linearity allowed quantification and identification of low levels of pesticides in the most difficult matrixes. The method has been used for routine analysis of many vegetables.     

Rapid determination of fumonisins in corn-based products by liquid chromatography/tandem mass spectrometry

A simple, fast, and robust method was developed for the determination of fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) in corn-based human food and animal feed (cornmeal). The method involves a single extraction step followed by centrifugation and filtration before analysis by ultra-performance liquid chromatographylelectrospray ionization (UPLC/ESI)-MS/MS. The LC/MS/MS method developed here represents the fastest and simplest procedure (<30 min) among both conventional HPLC methods and other LC/MS methods using SPE cleanup. The potential for high throughput analysis makes the method particularly beneficial for regulatory agencies and analytical laboratories with a high sample volume. A single-laboratory validation was conducted by testing three different spiking levels (200, 500, and 1000 ng/g for FB1 and FB2; 100, 250, and 500 ng/g for FB3) for accuracy and precision. Recoveries of FB1 ranged from 93 to 98% with RSD values of 3-8%. Recoveries of FB2 ranged from 104 to 108%, with RSD values of 2-6%. Recoveries of FB3 ranged from 94 to 108%, with RSD values of 2-5%.     

Rapid simultaneous determination of 14 sulfonamides in wastewater by liquid chromatography tandem mass spectrometry

This paper describes a rapid method for the determination of 14 kinds of sulfonamides (SAs) in wastewater using SPE, and LC-MS/MS with positive ESI (ESI(+)) and selected reaction monitoring (SRM) mode. The SPE was performed on an Oasis hydrophilic-lipophilic-balanced (HLB) cartridge. Chromatographic separation on a C18 column was achieved using a binary eluent containing methanol and water with 0.2% formic acid. Typical recoveries of the analytes ranged from 22.3 to 87.0% at a fortification level of 100 ng/L. The LODs in wastewater except sulfathiazole (3 ng/L) could be detected and quantified at levels as low as 1 ng/L. Finally, the method was applied to water from the municipal outlet and the aquaculture wastewater effluent. Sulfamethazine (SM(2)), sulfamethoxypyridazine (SMP), and sulfamethoxazole (SMZ) were most frequently found in wastewater in a concentration range between 1.2 and 31.7 ng/L.     

Quantitative determination of sparfloxacin in rat plasma by liquid chromatography/tandem mass spectrometry

Sparfloxacin, a fluoroquinolone antibiotic, is used for the treatment of bacterial infection. A quantification method using mass spectrometry was developed for the determination of sparfloxacin in rat plasma. After simple protein precipitation with acetonitrile, the analytes were chromatographed on a reversed‐phase C₁₈ column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma sparfloxacin concentrations after a single oral administration of sparfloxacin in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Fast determination of herbicides in waters by ultra-performance liquid chromatography/tandem mass spectrometry

A rapid multiresidue method for the analysis of more than 40 herbicides (such as simazine, terbuthylazine and diuron) in waters has been developed and validated by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS). Prior to chromatographic determination, the samples were extracted using a solid-phase extraction procedure. The analysis was performed on an Acquity UPLC BEH C(18) column using a gradient elution profile and a mobile phase consisting of methanol and an aqueous solution of formic acid (0.01%). Other chromatographic and MS/MS parameters were optimised in order to improve selectivity and sensitivity of the analytes. The analytes were detected using electrospray ionisation (ESI)-MS/MS in positive ion mode with multiple reaction monitoring (MRM), optimising parameters such as voltage cone, capillary voltage, source and desolvation temperature, and desolvation and cone gas flow. The optimised method provides a rapid separation (less than 10 min) of the selected herbicides in the assayed matrices, and it was validated by the analysis of spiked blank matrix samples. Good linearity was obtained and the repeatability of the method was less than 20% for the lowest calibration point. The limits of detection ranged from 0.002 to 0.02 microg/L, and the limits of quantification from 0.005 to 0.05 microg/L, which were below the values specified by the European Union. Finally, the method was successfully applied to real environmental samples from Andalusia (southern Spain). Terbuthylazine, simazine, atrazine desisopropyl and desethyl terbuthylazine were the herbicides most frequently found in water samples.     

Rapid determination of nitroimidazole residues in honey by liquid chromatography/tandem mass spectrometry

We developed a rapid and efficient means of determining residues of four nitroimidazoles-i.e., dimetridazole, ipronidazole, metronidazole, and ronidazole-and three hydrophilic metabolites- i.e., 2-hydroxymethyl-1-methyl-5-nitroimidazole, 1 -methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole, and 1-(2-hydroxyethyl)-2-hydroxymethyl-nitroimidazole--in honey. We applied a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure improved to suit a nitroimidazole analysis, which is fast (approximately 30 min) and uses less organic solvent. The procedure involves initial single-phase extraction of 5 g of honey with acetonitrile containing 1% acetic acid, followed by liquid-liquid partitioning involving the addition of 5 g sodium chloride, 1.5 g trisodium citrate dihydrate, and 4 g magnesium sulfate. Moreover, matrix from honey was reduced by an SPE method with an alumina-N cartridge. The samples were analyzed using LC/MS/MS. Chromatographic separation of these nitroimidazoles and metabolites was performed in the gradient mode on a pentafluorophenylpropyl-bonded silica column (150x2.0 mm, 3 pm particle size) at 40 degrees C. The mobile phase consisted of a 0.01% acetic acid solution and acetonitrile, and the flow rate was 0.2 mL/min. The method was validated using honey spiked with these nitroimidazoles from 0.1 to 0.5 microg/kg. The overall recovery of the seven nitroimidazoles ranged from 76.1 to 98.5%; intra- and interassay CV values were <9.5 and <14.2%, respectively. The LOQ ranged from 0.1 to 0.5 microg/kg. LC/MS/MS coupled with the QuEChERS method showed good potential as a method for determining nitroimidazole residues in honey.     

Characterization of titanocenium and titanocene by tandem mass spectrometry

Tandem mass spectrometric techniques were used for the characterization of gas-phase titanocenium ions. Decomposition of metastable and collisionally activated C₁₀H₁₀Ti^+˙ ions involved cyclopentadienyl–metal bond rupture, acetylene loss and dehydrogenation as the prominent processes. The intermediate formation of titanium (di)hydride complexes was proposed to explain the selective H₂ molecule loss. The neutralization–reionization mass spectrum showed a very abundant recovery signal, indicating a high stability for the neutral gas-phase C₁₀H₁₀Ti species.     

Simultaneous determination of 16 sulfonamides in honey by liquid chromatography/tandem mass spectrometry

A method is described for the determination of 16 sulfonamides in honey. Samples are dissolved in phosphoric acid solution (pH2), cleaned up with 2 solid-phase extraction (SPE) cartridges, an aromatic sulfonic cation-exchange cartridge and an Oasis HLB SPE cartridge, and analyzed both qualitatively and quantitatively by liquid chromatography/tandem mass spectrometry (LC/MS/MS) under the selected conditions. Without exception, calibration curves were linear (r = > 0.995), when sulfamethizole was between 1.0 and 25.0 microg/kg; sulfacetamide, sulfapyridine, sulfadiazine, sulfachloropyridazine, sulfamethoxazole, sulfamerazine, sulfisoxazole, sulfamonomethoxine, and sulfadoxine were between 2.0 and 50.0 microg/kg; sulfamethoxypyridazine, sulfadimethoxine, and sulfathiazole were between 4.0 and 100.0 microg/kg; sulfamethazine and sulfameter were between 8.0 and 200.0 microg/kg; and sulfaphenazole was between 12.0 and 300.0 microg/kg. Average recoveries at 4 fortification levels in the range of 1.0-300 microg/kg in honey were 70.9-102.5%, and relative standard deviations were 2.02-11.52%. The limits of quantitation for the 16 sulfonamides were between 1.0 and 12.0 microg/kg, with the LC/MS/MS method.     

The determination of melamine in muscle tissue by liquid chromatography/tandem mass spectrometry

In early 2007 it was determined that the compound melamine, suspected of having been involved in the deaths of numerous pets, had been fed to hogs intended for human consumption. This report describes a method for the analysis of melamine in porcine muscle tissue using solid-phase extraction (SPE) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Melamine was extracted in 50% acetonitrile in water. Homogenates were centrifuged and supernatants were acidified and washed with methylene chloride. The aqueous extracts were cleaned up using mixed-mode C8/strong cation exchange SPE and then concentrated, fortified with a stable isotope-labeled analog of melamine, and analyzed by HPLC/MS/MS. Gradient HPLC separation was performed using an ether-linked phenyl column with ammonium acetate/acetic acid and acetonitrile as the mobile phase. Multiple reaction monitoring (MRM) mode of two precursor-product ion transitions for melamine and one for the internal standard was used. A five point calibration curve ranging from 50 to 2000 ng/mL of melamine in solvent was used to establish instrument response. The method was validated by analysis of seven replicate porcine muscle tissue samples fortified with 10 ng/g of melamine. The mean recovery for the seven replicates was 83% with 6.5% relative standard deviation and the calculated method detection limit was 1.7 ng/g.