Liquid Chromatography-tandem Mass Spectrometric Analysis Research Articles

Quantitative liquid chromatography–tandem mass spectrometric analysis of 11-dehydroTXB2 in urine

A simple and selective determination method of 11-dehydrothromboxane B 2 (11-dehydroTXB 2 ), which is urinary metabolite of TXA 2 , has been developed employing liquid chromatography–tandem mass spectrometry (LC–MS–MS). 11-DehydroTXB 2 and its deuterium-labeled analogue as an internal standard were extracted from urine by simple solid-phase extraction (SPE). These compounds were analyzed using LC–MS–MS in the selected reaction monitoring (SRM) mode, by monitoring the transitions from m / z 367 to m / z 161 for 11-dehydroTXB 2 and from m / z 371 to m / z 165 for its internal standard. A good linear response over the range 50 pg–10 ng per tube was demonstrated. The values determined by LC–MS–MS were well validated and closely corresponded to the values determined by gas chromatography–mass spectrometry (GC–MS). The mean concentration of 11-dehydroTXB 2 in urine of healthy adults was 635±427 pg/mg creatinine (mean±S.D., n =13). This simple, accurate and selective determination method described in this study should greatly aid in evaluating the role of TXA 2 in vivo.

Quantitative liquid chromatography–tandem mass spectrometric analysis of 11-dehydroTXB 2 in urine

A simple and selective determination method of 11-dehydrothromboxane B 2 (11-dehydroTXB 2), which is urinary metabolite of TXA 2, has been developed employing liquid chromatography–tandem mass spectrometry (LC–MS–MS). 11-DehydroTXB 2 and its deuterium-labeled analogue as an internal standard were extracted from urine by simple solid-phase extraction (SPE). These compounds were analyzed using LC–MS–MS in the selected reaction monitoring (SRM) mode, by monitoring the transitions from m/ z 367 to m/ z 161 for 11-dehydroTXB 2 and from m/ z 371 to m/ z 165 for its internal standard. A good linear response over the range 50 pg–10 ng per tube was demonstrated. The values determined by LC–MS–MS were well validated and closely corresponded to the values determined by gas chromatography–mass spectrometry (GC–MS). The mean concentration of 11-dehydroTXB 2 in urine of healthy adults was 635±427 pg/mg creatinine (mean±S.D., n=13). This simple, accurate and selective determination method described in this study should greatly aid in evaluating the role of TXA 2 in vivo.

Proteomic Analysis of Glycosylphosphatidylinositol-anchored Membrane Proteins

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a functionally and structurally diverse family of post-translationally modified membrane proteins found mostly in the outer leaflet of the plasma membrane in a variety of eukaryotic cells. Although the general role of GPI-APs remains unclear, they have attracted attention because they act as enzymes and receptors in cell adhesion, differentiation, and host-pathogen interactions. GPI-APs may represent potential diagnostic and therapeutic targets in humans and are interesting in plant biotechnology because of their key role in root development. We here present a general mass spectrometry-based proteomic "shave-and-conquer" strategy that specifically targets GPI-APs. Using a combination of biochemical methods, mass spectrometry, and computational sequence analysis we identified six GPI-APs in a Homo sapiens lipid raft-enriched fraction and 44 GPI-APs in an Arabidopsis thaliana membrane preparation, representing the largest experimental dataset of GPI-anchored proteins to date.

High-throughput functional affinity purification of mannose binding proteins from Oryza sativa.

We have used affinity chromatography in combination with mass spectrometry to isolate, identify, and assign a preliminary functional annotation to a large number of both known and novel proteins from rice. Rice (Oryza sativa) leaf, root, and seed tissue extracts were fractionated by column affinity chromatography using alpha-D-mannose as the ligand. Bound fractions were eluted and subjected to one-dimensional electrophoresis, followed by high-performance liquid chromatography-tandem mass spectrometric analysis of separated proteins. This multiplexed technology resulted in the isolation and identification of 136 distinct mannose binding proteins from rice. A comparative analysis demonstrates very little overlap of identified proteins between the respective tissues, and confirms the correctly compartmentalized presence of a significant number of proteins from largely tissue-specific biochemical pathways. Over 30% of the identified proteins with a previously annotated function are directly involved in sugar metabolism, including several highly expressed known rice lectins. Direct comparison of the peptide sequences identified in this study to those peptides identified in the most comprehensive survey of the rice proteome to date indicates that our current data represents a significant enrichment of proteins unique to this dataset. Nearly 15% of the identified proteins, identified on the basis of exact peptide matching to sequences in the rice genomic database, represent proteins without a previously known functional annotation, indicating the potential of this combined chromatographic approach to assign a preliminary function to novel proteins in a high-throughput fashion.

Proteomic Consequences of a Human Mitochondrial tRNA Mutation beyond the Frame of Mitochondrial Translation

Numerous severe neurodegenerative and neuromuscular disorders, characterized biochemically by strong perturbations in energy metabolism, are correlated with single point mutations in mitochondrial genes coding for transfer RNAs. Initial comparative proteomics performed on wild-type and Myoclonic Epilepsy and Ragged Red Fibers (MERRF) mitochondria from sibling human cybrid cell lines revealed the potential of this approach. Here a quantitative analysis of several hundred silver-stained spots separated by two-dimensional gel electrophoresis was performed in the specific case of a couple of mitochondria, containing or not mutation A8344G in the gene for mitochondrial tRNALys, correlated with MERRF syndrome. Computer-assisted analysis allowed us to detect 38 spots with significant quantitative variations, of which 20 could be assigned by mass spectrometry. These include nuclear encoded proteins located in mitochondria such as respiratory chain subunits, metabolic enzymes, a protein of the mitochondrial translation machinery, and cytosolic contaminants. Furthermore, Western blotting combined with mass spectrometry revealed the occurrence of numerous isoforms of pyruvate dehydrogenase subunits, with subtle changes in post-translational modifications. This comparative proteomic approach gives the first insight for nuclear encoded proteins that undergo the largest quantitative changes, and pinpoints new potential molecular partners involved in the cascade of events that connect genotype to phenotype.

Liquid chromatography–tandem mass spectrometric analysis of surface and waste water with atmospheric pressure chemical ionisation: II. Applications

In Part I the design of the APCI interface and the scan modes of a triple quadrupole mass spectrometer have been described and general rules on how to use them have been discussed. Part II deals with the application of LC–APCI–MS/MS in the analysis of surface water and waste water. The goal is to analyse as many compounds as possible in one run. First, attention is paid to the LC part of the system to provide a good fit with the APCI interface, and the application and settings of the scan modes available. Next, results from the methods developed are shown, and matters such as their sensitivity and applicability are discussed. Finally, a CI–CID spectrum library concept is presented and discussed, which aims to solve the problem of the identification of unknown compounds.

Liquid chromatography–tandem mass spectrometric analysis of surface and waste water with atmospheric pressure chemical ionisation I: instrumentation

Liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS), i.e. LC–MS/MS, is a technique for the analysis of medium polarity to ionic compounds. This and the following paper highlight the potential and limitations of atmospheric pressure chemical ionisation (APCI) combined with MS/MS in the analysis of water. Part I describes the APCI interface and the scan modes of a triple quadrupole mass spectrometer. It gives a general introduction to the instrumentation, from a practical point of view. Part II gives more detailed information, in which the instrumentation is optimised for and used in the analysis of surface water and waste water. Attention is paid to the use of methanol versus acetonitrile as organic modifier in combination with APCI, the application of the RF-only daughter scan mode, and the quantitation of known compounds and the identification of unknown compounds found in surface and waste water.

Identification of losartan degradates in stressed tablets by LC-MS and LC-MS/MS

Three unknown peaks were observed in the severely stressed losartan tablets (at 40°C and 75% relative humidity for 3 years) analyzed by a stability indicating HPLC method. The sample solutions were subjected to liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis to obtain the chemical identities of these potential degradates. High accuracy and sensitivity of losartan and its degradates were obtained by using atmospheric pressure chemical ionization (APCI) technique in the LC-MS/MS analysis. Three trace level degradates (≤0.1%) were found to be the aldehyde and dimeric derivatives of losartan. The structural assignment was further confirmed by comparing the tandem mass spectra of the unknowns with those of the authentic materials of each corresponding degradate. Finally, the mechanistic pathways for the formation of the dimers are discussed.

Direct plasma liquid chromatographic-tandem mass spectrometric analysis of granisetron and its 7-hydroxy metabolite utilizing internal surface reversed-phase guard columns and automated column switching devices

An alternative on-line automated sample enrichment technique useful for the direct determination of various drugs and their metabolites in plasma is described for rapid development of highly sensitive and selective liquid chromatographic methods using mass spectrometric detection. The method involves direct injection of plasma onto an internal surface reversed-phase (ISRP) guard column, washing the proteins from the column to waste with aqueous acetonitrile, and backflushing the analytes onto a reversed-phase octyl silica column using switching valves. The analytes were detected using a tandem mass spectrometer operated in selected reaction monitoring (SRM) mode using atmospheric pressure chemical ionization (APCI). Use of two ISRP guard columns in parallel configuration allowed alternate injections of plasma samples on these columns for sample enrichment and shortened the column equilibration and LCMSMS analysis times, thereby increasing the sample throughput. The total run time, including both sample enrichment and chromatography, was about 6 min. Using this technique, an analytical method was developed for the quantitation of granisetron and its active 7-hydroxy metabolite in dog plasma. Granisetron is a selective 5-HT3 receptor antagonist used in the prevention and treatment of cytostatic induced nausea and vomiting. Recovery of the analytes was quantitative and the method displayed excellent linearity over the concentration ranges tested. Results from a three day validation study for both compounds demonstrated excellent precision (1.3–8.7%) and accuracy (93–105%) across the calibration range of 0.1 to 50 ng/ml using an 80 μl plasma sample. The automated method described here was simple, reliable and economical. This on-line approach using ISRP columns and column switching with LCMSMS is applicable for the quantification of other pharmaceuticals in pharmacokinetics studies in animals and humans which require high sensitivity.

Involvement of a serine protease in the synthesis of platelet-activating factor by endothelial cells stimulated by tumor necrosis factor-alpha or interleukin-1 alpha.

It has been shown that production of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells (EC) stimulated with tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha requires the synthesis of new proteins and is regulated by anti-proteinases. Here, we demonstrate that TNF-alpha and IL-1 alpha induce the expression by EC of a 34-kDa diisopropyl fluorophosphate-binding protein immunoprecipitated by an anti-human elastase antibody. This protein is released in the medium and cleaves the chromogenic substrate N-methoxysuccinyl- Ala-Ala-Pro-Val p-anilide, which is specific for elastase. The generation of this elastase-like protein seems to be important for the synthesis of PAF induced by TNF-alpha and IL-1 alpha, as suggested by the following observations: (a) it precedes the synthesis of PAF; (b) the inhibitors of serine protease and anti-human elastase antibody prevent the synthesis of PAF and the activation of 1-O-alkyl-2-lyso-glycerophosphocholine acetyl-CoA: acetyltransferase, which is a key enzyme of the PAF remodelling pathway; (c) elastase, at concentrations similar to that detectable in the medium of cytokine-activated EC, elicits a rapid synthesis of PAF by EC. High-performance liquid chromatography-tandem mass spectrometric analysis of bioactive PAF demonstrates that the molecular species produced after stimulation of EC with TNF-alpha, IL-1 alpha or elastase are similar, with a predominant synthesis of the alkyl species. These results indicate that TNF-alpha and IL-1 alpha stimulate the production of a serine protease which is critical in the activation of enzymes involved in PAF synthesis, suggesting the potential involvement of this mechanism in the regulation of EC functions.