Positive Electrospray Ionization Mass Spectrometry Research Articles

Simultaneous Quantification of Anandamide and Other Endocannabinoids in Dorsal Vagal Complex of Rat Brainstem by LC–MS

A quantitative method has been developed and validated for the simultaneous determination of anandamide (AEA), docosatetraenylethanolamide (DEA) and N-arachidonyldopamine (NADA) in dorsal vagal complex (DVC) of rat brainstem by liquid chromatographic-electrospray ionization mass spectrometry. The analytes were extracted from the tissue samples of rat brainstem by a single step liquid extraction technique using acetonitrile. The chromatographic separation was conducted on a C18 column using a gradient mobile phase consisting of methanol and water at a flow rate of 0.3 mL min(-1). The analytes were quantified by positive electrospray ionization mass spectrometry with selected ion monitoring (SIM) mode. The limits of detection (LOD) for AEA, DEA and NADA were 0.5, 1 and 0.5 ng mL(-1), respectively. This method required only simple processing of the samples and could be applied to monitor the change in the level of these compounds in DVC of the rat brain tissue. Time dependent (10-70 min) accumulation of the endocannabinoids (AEA, DEA, and NADA) in brain tissue was also studied, which included a novel examination of the accumulation of DEA as a function of time in rat brain tissue after decapitation.

Synthesis and characterization of the [Ru3O(CH3COO)6(py)2(BPE)Ru(bpy)2Cl](PF6)2 dimer

The polymetallic [Ru3O(CH3COO)6(py)2(BPE)Ru(bpy)2Cl](PF6)2 complex (bpy = 2,2′-bipyridine, BPE = trans-1,2-bis(4-pyridil)ethylene and py = pyridine) was assembled by the combination of an electroactive [Ru3O] moiety with a [Ru(bpy)2(BPE)Cl] photoactive centre, and its structure was determined using positive ion electrospray (ESI-MS) and tandem mass (ESI-MS/MS) spectrometry. The [Ru3O(CH3COO)6(py)2(BPE)Ru(bpy)2Cl]2+ doubly charged ion of m/z 732 was mass-selected and subject to 15 eV collision-induced dissociation, leading to a specific dissociation pattern, diagnostic of the complex structure. The electronic spectra display broad bands at 409, 491 and 692 nm ascribed to the [Ru(bpy)2(BPE)] charge-transfer bands and to the [Ru3O] internal cluster transitions. The cyclic voltammetry shows five reversible waves at −1.07 V, 0.13 V, 1.17 V, 2.91 V and −1.29 V (vs SHE) assigned to the [Ru3O]−1/0/+1/+2/+3 and to the bpy0/−1 redox processes; also a wave is observed at 0.96 V, assigned to the Ru+2/+3 pair. Despite the conjugated BPE bridge, the electrochemical and spectroelectrochemical results indicate only a weak coupling through the π-system, and preliminary photophysical essays showed the compound decomposes under visible light irradiation.

The fallaxidin peptides from the skin secretion of the Eastern Dwarf Tree Frog Litoria fallax. Sequence determination by positive and negative ion electrospray mass spectrometry: antimicrobial activity and cDNA cloning of the fallaxidins

The glandular skin secretion of the Eastern Dwarf Tree Frog Litoria fallax contains nine peptides named fallaxidins. The sequences of these peptides were elucidated using a combination of positive and negative electrospray mass spectrometry together with Edman sequencing. Among these peptides are: (i) fallaxidins 1.1 and 2.1 which have the sequences YFPIPI-NH2 and FWPFM-NH2. The activities of these peptides are unknown, but it has been shown that they are not smooth muscle active, opioids or antimicrobially active, nor do they effect proliferation of lymphocytes; (ii) two weakly active antibiotics, fallaxidins 3.1 and 3.2 (e.g. fallaxidin 3.1, GLLDLAKHVIGIASKL-NH2), and a moderately active antibiotic fallaxidin 4.1 (GLLSFLPKVIGVIGHLIHPPS-OH). Fallaxidin 4.1 has an unusual sequence for an antibiotic, containing three Pro residues together with a C-terminal CO2H group. cDNA cloning has confirmed the identity of the nine isolated peptides from L. fallax, together with five additional peptides not detected in the peptide profile. The pre-regions of the nine preprofallaxidins are conserved and similar to those of the caerin peptides from L. caerulea and L. splendida, suggesting that the fallaxidin and caerin peptides, although significantly different in sequence, originated from a common ancestor gene.

Antioxidant Pathways and One‐Electron Oxidation of Dopamine and Cysteine in Electrospray and On‐Line Electrochemistry Electrospray Ionization Mass Spectrometry

AbstractDopamine [DA]+ (m/z 154), DA dimer [2DA‐H]+ (m/z 307) and DA quinone [DAQ]+ (m/z 152) are detected in positive ion mode electrospray ionization mass spectrometry (ESI MS) of dopamine in 50/1/49 (vol%) water/acetic acid/methanol. H/D exchange experiments support a covalent structure of DA dimer. Thus, ESI of DA may involve 1e−, 1H+ oxidation processes followed by rapid radical dimerization. The DA quinone signal is low in ESI MS, which indicates a low efficiency of the 2e−, 2H+ oxidation reaction. On‐line electrochemistry ESI MS (EC/ESI MS) with low electrochemical cell voltage floated on high ES voltage increases electrospray current and improves sensitivity for DA. The DA quinone signal increases and DA dimer signal decreases. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity of ESI and EC/ESI MS measurements. A DA quinone‐cysteine adduct [DAQ+Cys]+ was detected in solutions of DA with cysteine (Cys). ESI MS and EC/ESI MS indicate formation of the DA quinone‐cysteine adduct by 1e− pathway. Oxidation pathways in ESI MS are relevant to biological reactivity of DA and Cys.

Determination of malachite green and leucomalachite green residues in water using liquid chromatography with visible and fluorescence detection and confirmation by tandem mass spectrometry

A liquid chromatography with visible and fluorescence detection (LC–vis/FLD) method for screening and a liquid chromatography with mass spectrometry (LC–MS/MS) method for the confirmation of malachite green (MG) and its major metabolite, leucomalachite green (LMG) residues in water have been described. Water samples were preconcentrated on diol solid-phase extraction columns. Chromatographic separation was achieved by using phenyl–hexyl column with an isocratic mobile phase consisting of acetonitrile and acetate buffer (0.05 M, pH 4.5) (70:30, v/v). In screening method liquid chromatography with absorbance detector was used for the detection of MG while LMG was detected by fluorescence detector. Detectors were connected on-line that allowed direct analysis of a sample extract for MG and LMG without the need of any post-column procedure. For the confirmation of MG and LMG in water positive electrospray ionization mass spectrometry in the multiple reaction monitoring mode was used. The developed methods have been validated according to the European Union requirements (Commission Decision 2002/657/EC). For LC–vis/FLD the mean recoveries at three fortification levels (0.4, 1, and 2 μg l −1) were in the range 95.4–104.7% for MG and 62.2–81.9% for LMG, whereas for LC–MS/MS, recoveries of MG and LMG were in the range 96.9–101.3% and 97.5–104.0%, respectively. Relative standard deviations of recoveries for both methods were less than 3.8 and 8.1% for MG and LMG, respectively. The stability of MG and LMG at 4 °C in darkness was observed for at least 10 months. Moreover, photo-oxidative decomposition products of analytes in water samples, observed in stability tests carried out at 20 °C, were identified by mass spectrometry as N-demethylated products of MG and LMG. These findings prove that N-demethylated products of MG and LMG, reported as potential carcinogens, may be formed in living fish organisms not only during enzymatic action but also during photo-oxidative degradation in water.

Quantification of Citalopram or Escitalopram and Their Demethylated Metabolites in Neonatal Hair Samples by Liquid Chromatography-Tandem Mass Spectrometry

Citalopram and escitalopram are highly selective serotonin reuptake inhibitors widely used in the treatment of depression. They exhibit adverse drug reactions and side effects, however, and the development of specific methods for their determination is of great interest in clinical and forensic toxicology. A liquid chromatography-tandem mass spectrometry method has been developed and validated for the assay of citalopram, escitalopram, and their demethylated metabolites in 10-mg hair samples. The analytes were extracted by incubation in methanol and liquid/liquid extraction with diethyl ether/dichloromethane. Gradient elution on a narrow bore C18 column was realized using clomipramine-d3 as an internal standard. Positive ion electrospray ionization and tandem mass spectrometry determination by collision-induced dissociation were performed in an ion trap mass spectrometer. The method exhibited a linear range of 25 to 2000 pg/mg, a quantification limit of 25 pg/mg for all analytes, relative standard deviations in the range of 12.10 to 9.80 (intraassay), and 13.80 to 11.78 (interassay), and accuracies (as percent recovery of the spiked standards) in the range of 90% to 110%; it was applied to the determination of citalopram and escitalopram and their metabolites in hair samples of two newborns to document their in utero exposure to the drugs. The method proved suitable for neonatal hair analysis of citalopram or escitalopram and was applied to two real cases of gestational exposure.

New features on the fragmentation and differentiation of C‐glycosidic flavone isomers by positive electrospray ionization and triple quadrupole mass spectrometry

Six flavone mono-C-glucosides, four standards (beta-D-glucopyranosyl-(1 --> C-6)- and -(1 --> C-8)- apigenin and luteolin) and two others from lemon juice (beta-D-glucopyranosyl-(1 --> C-6)- and -(1 --> C-8)-diosmetin) have been studied in order to analyze their fragmentation patterns. Initial separation was carried out using high-performance liquid chromatography with diode-array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer. Several systematic differences between collision-induced dissociation tandem mass (CID-MS/MS) spectra of C-6- and C-8-isomers have been found and some general guidelines and two new diagnostic product ions have been proposed for the differentiation of C-6- and C-8-flavonoid glycosides. These results have been successfully applied to the characterization of two flavone C-glycosides found in lemon juice, and mass spectra of a flavone di-C-glycoside detected in lemon juice have been studied and interpreted.

LC–MS analysis of trimethoxyamphetamine designer drugs (TMA series) from urine samples

A sensitive liquid chromatography–mass spectrometric (LC–MS) method for quantification of an active psychedelic hallucinogenic drugs (trimethoxyamphetamines) in human urine after solid-phase extraction (SPE) with C 18 cartridge was developed and validated. Chromatographic separation was achieved on reversed-phase Phenomenex 3.0 μm Polar Plus column (150 mm × 2.1 mm) with acetonitrile −0.2% acetic acid as mobile-phase and the step gradient elution resulted in a total run time of about 20 min. The analytes were detected by using an electrospray positive ionization mass spectrometry in selected ion monitoring (SIM) mode. In the evaluated concentration range (10–200 ng/mL) ( R 2 ≥ 0.998) a good linear relationship was obtained. The lower limits of detection (LLODs) and quantification (LLOQs) ranged from 4.26 to 9.12 ng/mL and from 13.18 to 29.22 ng/mL, respectively. Average recoveries ranged from 68.52 to 97.90% in urine at the concentrations of 25, 50 and 100 ng/mL. Intra- and inter-day relative standard deviations were 3.70–10.77% and 7.63–12.94%, respectively. This LC–MS method proved to be robust and reliable, and suitable for the use as a confirmation method in clinical urine drug testing.

Investigation by Mass Spectrometry of Metal Complexes of New Molecular Hosts: Cyclic Oligomer of Sugar Amino Acid and Sugar-Aza-Crown Ethers

The affinity of cyclic oligomers of sugar amino acid and sugar-aza-crown ether compounds towards various transition metal cations (Cu(II), Ni(II), Co(II), Fe(II) and Zn(II)) was investigated with positive-ion electrospray mass spectrometry. The binding between the receptors (M) and the different metals (Met) is evidenced mainly by the presence of the [M + Met(II)Cl](+) ion. The experimental results showed that all studied receptors present specificity to Cu(II). An attempt has been made with CuI but no complexation was obtained. The formation of these complexes can be rationalized by considering the presence of two oxygens and two nitrogens on the receptor rim. The lone electron pair can serve as the electron donor to Cu(II). Theoretical calculations were carried out in order to show the structure of the complex and, in particular, to determine if Cu(2+) is situated either on the outer surface, on the rim of the receptor or inside the cavity. Comparison of complex formation was carried out by mixing the four receptors with various amounts of Cu(II) (one equivalent and five equivalents). It appears that the best complexation was obtained with the sugar-aza-crown ethers (amine linker) for both benzylated and methylated compounds. In addition, the stereochemical effects have been investigated.

Positive‐ion ESI mass spectrometry of regioisomeric nonreducing oligosaccharide fatty acid monoesters: In‐source fragmentation of sodium adducts

Structural characterization and differentiation of a novel group of regioisomeric monolaurate esters of the nonreducing trisaccharides raffinose and melezitose, and the nonreducing tetrasaccharide stachyose has been obtained using positive electrospray ionization (ESI) mass spectrometry with in-source fragmentation. The surfactant nature and high polarity of these compounds make them appropriate analytes for being studied by conventional ESI-MS. The position of the acyl chain in each particular regioisomer has been used as a reporter group that allows unambiguous rationalization of the fragmentation routes of the corresponding natriated molecular ions [M + Na](+). In all cases, the main fragment ions were produced through cleavage of the glycosidic bond involving two anomeric carbons, the C-1' and C-2'' of the alpha-D-Glcp-(1-2)-beta-D-Fruf bond, and it was observed that sodium cation retention occurred on the heavier mass fragment of the two formed fragments, (e.g. di- or trisaccharide type vs monosaccharide type). Our results may help to better understand the fragmentation behavior of nonreducing oligosaccharides (as sodium adducts) in positive ESI mass spectrometry.

Determination of caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside in rat plasma using liquid chromatography–tandem mass spectrometry

A selective, sensitive and rapid liquid chromatography-tandem mass spectrometry method has been developed for the determination of caudatin-2,6-dideoxy-3-O-methy-beta-d-cymaropyranoside (CDMC) in rat plasma. This method involves a plasma clean-up step using liquid-liquid extraction, followed by LC separation and positive electrospray ionization mass spectrometry detection (LC/ESI-MS/MS). Chromatographic separation of the analytes was achieved using a C(18) column with a mobile phase of acetonitrile and water (70:30, v/v) at a flow rate of 1.0 mL/min. Low energy collision tandem mass spectrometric analysis (CID-MS/MS) using the multiple reaction monitoring (MRM) mode was used for analyte quantification. For the MRM analysis of CDMC, the following transition at m/z 658.4 --> 529.6 derived from the protonated molecule [M + Na](+). A calibration curve was linear in the 5-500 ng/mL range for CDMC, and the limit of detection was 5 ng/mL. The inter- and intra-day precisions (RSD) were <or=8.5 and <or=9.1%, respectively. The accuracy (RE) was +/- 10.9%. The novel developed LC/ESI-MS/MS method was successfully applied to pharmacokinetic studies of CDMC after oral administration to rats. The total chromatographic run time of this method was 5.0 min per sample, allowing for analysis of a large number of samples per batch.

Simultaneous determination of rifampicin and levofloxacin concentrations in catheter segments from a mouse model of a device-related infection by liquid chromatography/electrospray ionization tandem mass spectrometry

The aim of this study was to develop a specific and sensitive liquid chromatography mass spectrometry (LC/MS) method for the determination of rifampicin and levofloxacin concentrations from infected tissues within teflon catheter segments which were subcutaneously implanted in mice. A solid-phase extraction procedure was used to extract analytes from tissue homogenates of the catheter segments and reverse-phase HPLC combined with positive electrospray ionization mass spectrometry was used for analyte separation and quantification. The assay was found to be linear over the concentration range of 0.02–2 μg/g for rifampicin and levofloxacin in tissues and provided good validation data for accuracy and precision. The intra-day accuracy as determined by the relative error was −1.3% for levofloxacin and 6.1% for rifampicin, and precision was evaluated by R.S.D.s with a maximum of 5.1% for levofloxacin and 8.1% for rifampicin. The inter-day accuracy was −3.3% for levofloxacin and −4.6% for rifampicin, and precision was 8.6% for levofloxacin and 7.1% for rifampicin. The assay uses less tissue than previously described methods and can be applied to determine the penetration of rifampicin and the fluoroquinolone in catheter segments from a mouse model of a device-related infection. Finally, the HPLC–MS assay should be applicable to studies of rifamycin + quinolone combination therapies in other animal models of bacterial infection.

Study on [(4‐substituted benzoylamino)phenylmethyl]phosphonic acid diisopropyl esters under electrospray ionization tandem mass spectrometric conditions

Phosphonate esters were synthesized and investigated by positive ion electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). It was found that for the sodiated adduct there were two novel rearrangement reactions, in which the phosphoryl oxygen atom migrated to the carbonyl carbon and a cleavage occurred at the amide bond, or a benzylamine fragment was lost and the phosphorus was reduced. However, when the methyl group substituted for the isopropyl group, these migrations were inhibited. A possible mechanism was proposed, and high-resolution mass spectrometry was applied to identify the formula of these novel ions. These results showed that ESI-MS is a useful tool for the structural determination of phosphonate esters.

Quantitative analysis of total retronecine esters-type pyrrolizidine alkaloids in plant by high performance liquid chromatography

Pyrrolizidine alkaloids (PAs) are alkaloids which typically contain a necine (7-hydroxy-1-hydroxymethyl-6,7-dihydro-5H-pyrrolizidine) base unit, and they can be found in one third of the higher plants around the world. They are hepatotoxic, mutagenic and carcinogenic and pose a threat to human health and safety. A specific, quick and sensitive method is therefore needed to detect and quantify the PAs sometimes in trace amount in herbs, tea or food products. Based on high performance liquid chromatography with prior derivatization of the alkaloids using o-chloranil and Ehrlich's reagent, we report an improved method for quantitative analysis of the total amount of retronecine esters-type pyrrolizidine alkaloids (RET-PAs) in a plant extract. The total quantitation of RET-PAs is achieved because of a common colored retronecine marker, a 7-ethoxy-1-ethoxylmethyl retronecine derivative, is produced with all the different RET-PAs during the derivatization reaction. The chemical identity of the common retronecine marker was characterized on-line by positive mode electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. The limit of detection using the improved method is 0.26 nmol mL −1 and the limit of quantitation is 0.79 nmol mL −1. The advantages of this method are much enhanced sensitivity in detection and quantitation, and, no restriction on the choice of RET-PA as a calibration standard. Application of the developed method to the quantitation of total RET esters-type PAs in Senecio scandens from different regions of China is also reported.

The cobalt–albumin binding assay: Insights into its mode of action

BackgroundWe previously hypothesized that the N-terminus of human serum albumin (HSA) is altered during ischemic events, thus establishing the foundation for the cobalt–HSA binding assay phenomenon. In this investigation, we attempt to clarify the mode of action of the cobalt–HSA binding assay by direct observations of cobalt binding to HSA. MethodsHigh pressure liquid chromatography coupled to positive electrospray ionization mass spectrometry (HPLC/MS) was used to study cobalt binding to HSA in the plasma of patients with and without evidence of myocardial ischemia. ResultsStrong binding of cobalt to the N-terminus of HSA occurs at pH>7.0. No differences in cobalt binding to the N-terminus of HSA are observed in ischemic versus non-ischemic patients' plasma despite differences in the cobalt–HSA binding assay. Plasma free cysteine/cystine ratio appears to play a role in the quantitative response of the cobalt–HSA binding assay. ConclusionsThe main determinants of the cobalt–HSA binding assay mechanism of action include but are not limited to: the proportion of intact N-terminus of HSA, HSA concentration, plasma cysteine/cystine ratio, plasma pH, and the state of oxidation of cys34 of HSA. Assay improvements that consider and take these factors into account could lead to an improved cobalt–HSA binding assay with greater clinical utility.

ESI-MS analyses of lake dissolved organic matter in light of supramolecular assembly

A high-performance size-exclusion chromatography (SEC) system was coupled on-line to an electrospray ionization (ESI) interface to detect gas-phase ions by an API 365 LC/MS/MS triple quadrupole analyzer. The SEC fractions of a strongly coloured freshwater solution containing dissolved organic matter-humic substances (DOM-HS) were screened both by UV(254) and by ESI mass spectrometry (ESI-MS) in the full-scan mode within the m/z range of 100-2,900 amu in negative and positive polarities. The ESI-MS spectra were also collected by direct infusion of the DOM-HS solution in both polarities. ESI-MS spectra did not primarily favour low mass compounds, and negative and positive total ion chromatograms were parallel to the SEC elution profile obtained by UV(254) detection from DOM-HS solution. The UV(254) detection overestimated the SEC portion of higher size/mass solutes and underestimated that of solutes of smaller sizes/masses as compared with the total ion chromatogram intensities in negative or positive polarities. The change of mass-weighted and number-weighted average sizes/masses (M(w) and M(n)) of different SEC fractions was fairly small, in contrast to UV(254) detection, with increasing elution volume. A reasonable explanation for the great differences between M(w) and M(n) values, obtained by UV(254) and ESI-MS detections for eight different SEC fractions, seems to be a supramolecular-type association of relatively small components through weak dispersive forces. M(n) values obtained by vapour-pressure osmometry for different SEC fractions were to some extent analogous with those of negative and positive ESI-MS. The shapes obtained by either negative or positive polarities and calculated M(w) and M(n) values indicated a close structural similarity between each SEC fraction. Positive ion and negative ion spectra of different humic fractions represented quite similar components, and there was no evidence for noteworthy occurrence of multiply charged ions being able to lower mass distributions of negative ion spectra. The effect of nitrogen on the mass spectra seemed to be unimportant, and the weak ions observed at even m/z values correspond most likely to the (13)C counterparts of the more abundant (12)C odd ions. No uncontrolled ESI fragmentation was observable and humic solutes seemed to be quite heat-resistant. Direct infusion of the untreated DOM-HS solution and statistical calculation verified that the SEC-separated different fractions really represent distinct entities of the original DOM-HS mixture. ESI-MS results support the opinion that the structural composition of humic solutes in their original combined mixture resembles supramolecular-type associations of smaller molecular size entities possessing similar structural functionalities.

Synthesis and Electrospray Ionization Mass Spectra of N‐(1,3, 2‐Dioxaphosphorinan‐2‐ylmethyl)thiophosphoramidates

AbstractN‐(1,3,2‐Dioxaphosphorinan‐2‐ylmethyl)thiophosphoramidates were synthesized and determined by NMR spectra and positive ion electrospray ionization mass spectrometry (ESI‐MS) in conjunction with tandem mass spectrometry (MS/MS). The fragmentation pathways were investigated. The results show that these characteristic ions in ESI mass spectra are useful in the structural determination of N‐(1,3,2‐dioxaphosphorinan‐2‐ylmethyl)thiophosphoramidates.

Fragmentation Study of Globularin Through Positive and Negative ESI/MS, CID/MS, and Tandem MS/MS

The structure of globularin was studied by a mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision‐induced dissociation (CID), and tandem mass spectrometry. The mass spectrometry investigation was achieved through in‐source fragmentation of the deprotonated [M−H]−, protonated [M+H]+, lithiated [M+Li]+, sodiated [M+Na]+, and potassium‐cationized [M+K]+ ions. This allowed collision‐induced dissociation spectra of the ionized molecular ions to be obtained to give valuable structural information regarding the nature of both the glycoside and the aglycone moieties and the effect of metal cationization on the CID spectra. Glycosidic fission and ring cleavages of both aglycone and sugar moieties were the major fragmentation pathways observed during collision‐induced dissociation, where the losses of small molecules, the cinnamoyl and the cinnamate parts were also observed. Alkali metal cationization offers additional fragmentation pathways involving cross rings cleavage under CID conditions. Unlike the dissociation of protonated molecular ions, that of metal‐cationized molecules also provides sugar fragments where the C0 + fragment corresponding with the glucose ion was obtained as a major daughter peak for all the studied compounds. Even with low abundance, fragment ions coordinated to K+ were also observed from [M+K]+.

Determination of caderofloxacin lactate in rat plasma by high-performance liquid chromatography–mass spectrometry and its application in rat pharmacokinetic studies

A sensitive liquid chromatography–electrospray ionization mass spectrometric (LC–ESI-MS) method for the quantification of a newly active quinolone carboxylic acid caderofloxacin lactate in rat plasma was developed and validated after precipitation method with methanol. Chromatographic separation was achieved on a reversed-phase Shimadzu 2.0 μm C18 column (150 mm × 2.00 mm) with the mobile phase of methanol–0.02% formic acid and step gradient elution resulted in a total run time of about 10.0 min. The analytes were detected by using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (5–2000 ng/mL) ( r = 0.9998). The lowest limit of quantification (LLOQ) was 5 ng/mL and the lowest limit of detection (LLOD) was 2 ng/mL. Average recoveries ranged from 88.80 to 93.05% in plasma at the concentrations of 10, 100 and 1000 ng/mL. Intra- and inter-day relative standard deviations were 4.01–7.30 and 4.15–7.51%, respectively. This method was successfully applied in the pharmacokinetic studies in rats.

FcϵRI and Thy-1 domains have unique protein and lipid compositions

Receptor activation leads to the dynamic remodeling of the plasma membrane. Previous work using immunoelectron microscopy showed that aggregated high-affinity receptor for immunoglobulin E (FcRI) and aggregated Thy-1, a glycerophosphoinositol (GPI)-anchored protein, have distinct membrane distributions. We now report lipidomics analysis of FcRI- and Thy-1-enriched vesicles obtained by magnetic bead isolation in the absence of detergent. Protein analyses show that FcRI domains are enriched in receptors and associated signaling molecules, whereas Thy-1 domains are devoid of FcRI subunits. Positive and negative ion electrospray mass spectrometry demonstrated that both domains retained a complex mixture of phospholipid classes and molecular species, predominantly glycerophosphocholine, glycerophosphoethanolamine (GPE), and sphingomyelin as well as glycerophosphoserine and GPI lipids. Analysis of total acyl groups showed that < 50% of fatty acids in these domains are fully saturated, inconsistent with the recruitment of aggregated receptors or GPI-anchored proteins to liquid ordered domains. However, further analysis showed that FcRI domains contain two times more sphingomyelin and a high ratio of cholesterol to total fatty acid content compared with Thy 1-enriched domains. Remarkably, plasmenyl glycerophosphoethanolamine phospholipids (plasmalogen GPE) were also 2.5-3 times more abundant in FcRI domains than in the Thy-1 microdomains, whereas most diacyl GPE molecular species were equally abundant in the two domains.