Positive Ion ESI Research Articles

Quantitative and Qualitative Determination of Liuwei Dihuang Tablets by HPLC-UV-MS-MS

A method is developed for the analysis of allantoin, gallic acid, dihydromelittoside, loganin, paeoniflorin, benzoylpaeoniflorin, and paeonol in Liuwei Dihuang tablets by high-performance liquid chromatography (HPLC)-UV-mass spectrometry (MS)-MS. Gradient elution with methanol-acetonitrile-water-formic acid solvent system is employed in the HPLC-electrospray ionization-MS study. The positive-ion ESI mode is suitable for these compounds. The peaks of gallic acid, loganin, dihydromelittoside, paeoniflorin, benzoylpaeoniflorin, and paeonol are identified by their mass spectra and the fragments of their MS-MS spectra. Allantoin, gallic acid, loganin, paeoniflorin, and paeonol are simultaneously determined by UV detection at 210 nm for quantitative purposes.

Effect of Protein Stabilization on Charge State Distribution in Positive- and Negative-Ion Electrospray Ionization Mass Spectra

Changes in protein conformation are thought to alter charge state distributions observed in electrospray ionization mass spectra (ESI-MS) of proteins. In most cases, this has been demonstrated by unfolding proteins through acidification of the solution. This methodology changes the properties of the solvent so that changes in the ESI-MS charge envelopes from conformational changes are difficult to separate from the effects of changing solvent on the ionization process. A novel strategy is presented enabling comparison of ESI mass spectra of a folded and partially unfolded protein of the same amino acid sequence subjected to the same experimental protocols and conditions. The N-terminal domain of the Escherichia coli DnaB protein was cyclized by in vivo formation of an amide bond between its N- and C-termini. The properties of this stabilized protein were compared with its linear counterpart. When the linear form was unfolded by decreasing pH, a charge envelope at lower m/z appeared consistent with the presence of a population of unfolded protein. This was observed in both positive-ion and negative-ion ESI mass spectra. Under the same conditions, this low m/z envelope was not present in the ESI mass spectrum of the stable cyclized form. The effects of changing the desolvation temperature in the ionization source of the Q-TOF mass spectrometer were also investigated. Increasing the desolvation temperature had little effect on positive-ion ESI mass spectra, but in negative-ion spectra, a charge envelope at lower m/z appeared, consistent with an increase in the abundance of unfolded protein molecules.

Electrospray ionization‐multistage tandem mass spectrometry of complex multitin organometallic compounds

The series of 14 complex organotin(IV) compounds containing many tin atoms and noncovalent bonds in the structure was characterized by electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). The mass spectra were measured in both polarity modes to obtain complementary structural information. The characteristic pattern of ten natural tin isotopes allowed the determination of the number of tin atoms in the molecular adducts and fragment ions by comparing theoretical and experimental isotopic distributions. Positive ion ESI spectra show unusual adduct formation depending on the type of organic solvent used for the direct infusion analysis owing to the ion-molecule reactions in the ion source. On the basis of the detailed spectral interpretation of organotin(IV) compounds, the fragmentation patterns of multitin organometallic compounds have been proposed. Noncovalent bonds in polymeric complexes are fragmented first, which is then followed by characteristic neutral losses in monomeric units.

Rapid separation and identification of phenolic and diterpenoid constituents from Radix Salvia miltiorrhizae by high‐performance liquid chromatography diode‐array detection, electrospray ionization time‐of‐flight mass spectrometry and electrospray ionization quadrupole ion trap mass spectrometry

High-performance liquid chromatography-diode array detection (HPLC-DAD), electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) and electrospray ionization quadrupole ion trap mass spectrometry (HPLC-ESI-MSn) were used for the isolation, identification and structural analysis of water-soluble phenolic and nonpolar diterpenoid constituents in Dan-shen (Radix Salvia miltiorrhizae) which was prepared by sonication in 70% methanol. Mass spectra were obtained by ESI-TOF-MS and electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS). A formula database of known constituents in Dan-shen was established and most constituents were rapidly identified by HPLC-DAD/ESI-TOF-MS by matching their accurate molecular masses with the formulae of the compounds in the database. Compounds with the same molecular formula could not be differentiated by TOF-MS; however, QIT-MS could differentiate those compounds and elucidate their structures based on their characteristic fragmentation. HPLC-DAD, HPLC/ESI-TOF-MS and HPLC/ESI-MSn provided complementary information for the identification of the constituents in Dan-shen. Forty constituents were identified in 30 min based on their positive and negative ion ESI mass spectra and liquid chromatographic information. Thus the method described is useful for the rapid analysis of multiple constituents in Dan-shen.

Imidazolium scaffolds: Electrospray mass spectrometric observation of N-heterocyclic carbenes in positive-ion mode and halide ion adducts in negative-ion mode

The gas-phase behavior of dicationic protophanes 3a·2X and [1 4]imidazoliophanes 4a·2X and 5a·2X were examined by electrospray mass spectrometry in both positive- and negative-ion mode. The neutral analytes underwent deprotonation and the positive-ion mode ESI experiments displayed N-heterocyclic carbenes (NHC) whereas the negative-ion mode response showed a variety of polymolecular self-assemblies due to non-covalent interactions with halide anions. Moreover, positive-ion ESI tandem mass spectrometry of dications 3a·2Cl and 4a·2Cl revealed the formation pathway of the carbene species; the neutral analytes M·2Cl produced loss of one counteranion followed by a neutral loss of HCl, which led to the deprotonated singly-charged imidazolylidene ion [M − H] +.

Characteristic Fragmentation Behavior of Steroidal Phosphor‐ amidate Conjugates in Electrospray Ionization Tandem Mass Spectrometry

AbstractNovel steroidal phosphoramidate conjugates of 3′‐azido‐2′,3′‐dideoxythymidine (AZT) and amino acid esters were synthesized and determined by positive and negative ion electrospray ionization mass spectrometry. The MS fragmentation behaviors of the steroidal phosphoramidate conjugates have been investigated in conjunction with tandem mass spectrometry of ESI‐MS/MS. There were three characteristic fragment ions in the positive ion ESI mass spectra, which were the Na adduct ions with loss of steroidal moiety, amino acid ester moiety from pseudo molecular ion (M+Na)+, and the phosphoamino acid methyl ester Na adduct ion by α‐cleavage of the phosphoramidate respectively. The main fragment ions in negative ion ESI mass spectra were the ion (M−HN3)−, the ion (M−AZT−H)−, and the ion (M−steroidal moiety−H)− besides the pseudo molecular ion (M−H)−. The fragmentation patterns did not depend on the attached amino acid ester moiety.

Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases

Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.

Potentials of ion trap collisional spectrometry for liquid chromatography/electrospray ionization tandem mass spectrometry determination of buprenorphine and nor‐buprenorphine in urine, blood and hair samples

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the analysis of buprenorphine (BUP) and nor-buprenorphine (NBUP) in biological fluids. Analytes are isolated from urine and blood, after addition of d4-buprenorphine (d4-BUP) as internal standard, by solid-phase extraction. Preparation of hair involves external decontamination, mechanical pulverization, overnight incubation in acidic medium, and neutralization prior to extraction. Enzymatic hydrolysis with beta-glucuronidase may be performed to distinguish between free and total BUP. Chromatographic separation is accomplished by gradient elution on a cyanopropyl 2.1 x 150 mm column. Positive ion ESI and MS analyses are carried out in an ion trap mass spectrometer. The use of this mass analyzer allows effective collisional experiments to be performed on ESI-generated MH+ species. Abundant product ions are produced, which can be monitored together with precursor ions without losing sensitivity. Thus, assay selectivity is definitely increased with respect to LC/ESI-MS/MS methods in which only precursor ions are monitored. The method has good linearity (calibration curves were linear in the range 0.1-10 ng/mL in urine and blood, in the range 10-160 pg/mg in hair) and limits of detection of 0.05 ng/mL for both BUP and NBUP in blood and urine samples, of 4 pg/mg for both analytes in hair. Both intra- and inter-assay precision and accuracy were satisfactory at three concentrations studied: relative standard deviations were <13.7% in urine, <17.3% in blood, <17.8% in hair; percent deviation of the mean from the true value was always <10.5% in urine and blood, <16.1% in hair. The method can be used to determine both analytes in the urine and hair of drug addicts on replacement therapy, and in post-mortem blood specimens when there is suspicion of drug-related death.

Use of Saturates/Aromatics/Resins/Asphaltenes (SARA) Fractionation To Determine Matrix Effects in Crude Oil Analysis by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

We have previously demonstrated the ability of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to resolve and identify the polar species found in all petroleum distillates. The ultrahigh resolving power and mass accuracy of FT-ICR MS allow for the identification of thousands of compounds in crude oils without prior chromatographic separation. Here, we compare positive-ion ESI FT-ICR mass spectra of a South American crude oil with spectra of its saturates/aromatics/resins/asphaltenes (SARA)-isolated asphaltenes, resins, and aromatics, to ascertain the effect of the other components on the relative mass spectral abundances of the polar aromatics. Saturates are unobservable by ESI. For the chosen oil, little to no signal was obtained for the asphaltenes and resins because of their mostly acidic nature. The mass distributions, heteroatom “class” distributions, “type” (rings plus double bonds) distributions, and carbon number distributions of the aromatic fra...

Mononuclear to Polynuclear Transition Induced by Ligand Coordination: Synthesis, X-ray Structure, and Properties of Mono-, Di-, and Polynuclear Copper(II) Complexes of Pyridyl-Containing Azo Ligands

Reactions of two hydrated cupric salts (CuCl(2).2H(2)O and Cu(ClO(4))(2).6H(2)O) with three azopyridyl ligands, viz. 2-[(arylamino)phenylazo]pyridine [aryl = phenyl (HL(1a)), p-tolyl (HL(1b)), and 2-thiomethyl phenyl (HL(1c))], 2-[2-(pyridylamino)phenylazo]pyridine (HL(2)), and 2-[3-(pyridylamino)phenylazo]pyridine (HL(3)), afford the mononuclear [CuClL(1)] (1), dinuclear [Cu(2)X(2)L(2)(2)](n)()(+) (X = Cl, H(2)O, ClO(4); n = 0, 1; 2, 3), and polynuclear [CuClL(3)](n)() (4) complexes, respectively, in high yields. Representative X-ray structures of these complexes 1-4 are reported. X-ray structure analysis of 4 reveals an infinite 1D zigzag chain that adopts a saw-tooth-like structure. Variable-temperature cryomagnetic measurements (2-300 K) on the complexes 2-4 have revealed weak magnetic interactions between the copper centers with J values -1.04, 9.88, and -1.31 cm(-1), respectively. Positive ion ESI mass spectra of the soluble complexes 1-3 are studied which provide the evidence for the integrity of the complexes also in solution. Visible range spectra of the complexes 1-3 in solution consist of intense and broad transitions in the range 700-600 nm. The solid-state spectrum of the insoluble copper complex 4, on the other hand, shows a structured band near 700 nm. The intensities of the transitions of the dinuclear complexes are much higher than those of the corresponding mononuclear copper complexes. Redox properties of the present copper complexes are reported. Notably, the dinuclear complex, 3, displays two successive redox processes: Cu(II)Cu(II) right harpoon over left harpoon Cu(II)Cu(I) right harpoon over left harpoon Cu(I)Cu(I). It catalyzes aerial oxidation of L-ascorbic acid. The catalytic cycle is most effective up to H(2)A/3 (H(2)A = L-ascorbic acid) molar ratio of 20:1.

Pharmacokinetic measurements of IDN 5390 using electrospray ionization tandem mass spectrometry: structure characterization and quantification in dog plasma

In this report, electrospray ionization tandem mass spectrometry (ESI-MS/MS) for a pharmacokinetic study of IDN 5390, a novel C-seco taxane derivative, which is under preclinical evaluation, has been investigated. Our results showed that IDN 5390 and other taxanes including paclitaxel and IDN 5109 could ionize well in not only positive-, but also in negative-ion mode. Under collision-induced dissociation (CID) conditions, these compounds could fragment into similar M- (molecular), T- (taxane ring) and S- (side chain) series ions. In positive-ion ESI, the formation of both T- and S-series ions involved the breaking of the C-13 ester bond. In negative-ion ESI, however, while the formation mechanism of S-series ions remained the same, the breaking of the C-1' carboxylic ester bond resulted in T-series ions. At optimum collision energy (CE) values, M-, T- and S-series ions of IDN 5390 in both positive- and negative-ion ESI-MS/MS spectra had good intensity. This phenomenon makes both positive- and negative-ion ESI-MS/MS good methods for IDN 5390 metabolite structural characterization, i.e. to reveal the location of modification groups in IDN 5390 metabolites versus IDN 5390 either on the side chain or the taxane ring. A liquid chromatography (LC)/ESI-MS/MS method using the multiple-reaction monitoring (MRM) technique was thereafter developed to quantify IDN 5390 in dog plasma using paclitaxel as internal standard. The method was validated using a concentration range between 5 and 1000 ng/mL and had a limit of detection of 1 ng/mL. The inter-day %CV (%coefficient of variation) of the calibration standards ranged between 4.36 and 9.64%, the intra-day %CV of the calibration standards between 0.61 and 13.44%, and the mean % accuracy of the quality control samples at the low, middle and high end of the concentration curves were 12.5, 6.8 and 9.6%, respectively.

Electrospray ionization and tandem mass spectrometry characterization of novel heterotrimetallic Ru(η 5-C 5H 5)(dppf)SnX 3 complexes and their heterobimetallic Ru(η 5-C 5H 5)(dppf)X precursors

Electrospray ionization and tandem mass spectrometry were used to characterize some novel heterotrimetallic complexes Ru(η 5-C 5H 5)(dppf)SnX 3 ( 1) (dppf = diphenylphosphine ferrocene; X = Cl, Br) and their heterobimetallic precursors Ru(η 5-C 5H 5)(dppf)X ( 2) (X = Cl, Br, I, N 3). The positive-ion ESI mass spectra of 1 and 2 in acetonitrile solution showed the predominant presence of the [Ru(η 5-C 5H 5)(dppf)] + cation ( 3) of m/ z 721. In the positive-ion ESI mass spectrum of the complex Ru(η 5-C 5H 5)(dppf)N 3 ( 2d), another prominent cation of m/ z 735, i.e. [Ru(η 5-C 5H 5)(dppf)N] + ( 4), was also observed. Its formation was proposed to occur via the oxidation of the ruthenium atom (Ru II → Ru III) followed by the loss of N 2. The positive-ion ESI tandem mass spectra of ions 3 and 4 showed mainly fragments formed by neutral losses of C 5H 5, C 6H 5 and P(C 6H 5) 2. Finally, cation 3 was proposed to be the catalytic active species in the single-step conversion of methanol to acetic acid.

Determination of testosterone in saliva and blow of bottlenose dolphins ( Tursiops truncatus) using liquid chromatography–mass spectrometry

A rapid, accurate and reproducible assay utilising high performance liquid chromatography–mass spectrometry (LC–MS) has been developed and validated for determining testosterone concentrations in saliva and blow of bottlenose dolphins. Sample preparation used solid phase extraction with specific preconditioning of cartridges. Analytes were eluted with 100% acetonitrile, dried under nitrogen and stored at −80 °C. Samples were reconstituted in 60% acetonitrile for LC–MS analysis. Chromatographic separation was achieved with an Alltech Macrosphere C8 stainless steel analytical column (2.1 mm × 150 mm i.d., 5 μm particle size, 300 Å pore size) using a 55% mobile phase B isocratic method (mobile phase A = 0.5% acetic acid; mobile phase B = 0.5% acetic acid, 90% acetonitrile). Samples were analysed in SIM at m/ z 289.20 (testosterone mw 288.40) and a positive ion ESI. The limit of quantification was 0.5 ng/ml with a limit of detection of 0.2 ng/ml. The concentration curve was linear from 0.5 to 50 ng/ml ( y = 0.01 x + 0.0045, r 2 = 0.959, r = 0.979, p < 0.001). The R.S.D.s of intra- and inter-batch precision were less than 15% for saliva and 11% blow. Recovery of the assay for saliva was 93.0 ± 7.9% (50 ng/ml) and 91.5 ± 3.72% (1 ng/ml), and for blow was 83.3 ± 6.8% (50 ng/ml) and 85.8 ± 4.6% (1 ng/ml). Recovery of the internal standard in saliva was 73.0 ± 14.2% and in blow was 78.63 ± 4.29. The described assay was used to determine the presence of endogenous testosterone in saliva (9.73–23 ng/ml, n = 10) and blow (14.71–86.20 ng/ml, n = 11) samples of captive bottlenose dolphins.

Rapid identification and quantitation of compounds with forensic interest using fast liquid chromatography–ion trap mass spectrometry and library searching

A fast liquid chromatography–electrospray tandem mass spectrometric (LC–ESI-MS–MS) method by using a monolithic column, gradient elution and ion trap mass spectrometer was developed for 14 forensically interesting and chemically different compounds. All compounds were eluted within 2.5 min and the total analysis time was 5 min including stabilisation time required for the next injection. All the compounds, basics, neutrals and acids were efficiently ionised by positive ion ESI. A laboratory library including MS–MS spectra and retention times was developed and tested. Results with 476 standard samples and 50 authentic samples showed that the compounds studied can be unambiguously identified with the library. A quantitative method was developed for the compounds using external calibration. The evaluation process showed good linearity of the method and reasonable repeatability. Limits of detection ranged from 10.0 to 50.0 ng/ml.

Carbohydrate Recognition in Cross-Linked Sugar-Templated Poly(acrylates)

A low reloading capacity is a serious weakness for any recognition material. To raise the overall amount of saccharides bound by templated poly(acrylates) with immobilized copper(II) complexes, I enlarged the number of attractive binding forces inside the matrix by polymerizing cross-linking monomers with various amounts of free hydroxyl groups. The material preparation was conducted in the presence of either aqueous methanol or alkaline water as porogenic solvent. All rebinding experiments were carried out in water at physiological pH. Positive ion ESI mass spectrometry shows only weak copper(II)−saccharide interactions under sugar rebinding conditions. The formation of multiple hydrogen bondings between the polymer backbone and the sugar template are demonstrated to be advantageous for the overall material capacity. Increasing the flexibility of the polymer backbone in a templated polar matrix enhances the overall material capacity for disaccharides (∼80 μmol of sugar per gram polymer) and diminishes the material selectivity only slightly. The polymer selectivity for discrimination of α- and β-glycosidic bonds in the disaccharides maltose and lactose is higher in a polar than in a nonpolar matrix. The polymer preference for the smaller, less polar monosaccharide glucose decreases along with an increasing matrix polarity in favor of the larger, more polar disaccharide lactose.

Determination of polycyclic aromatic hydrocarbons by liquid chromatography–electrospray ionization mass spectrometry using silver nitrate as a post-column reagent

Liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) using silver nitrate as a post-column reagent has been used for the determination of 10 polycyclic aromatic hydrocarbons (PAHs) in river water. In this method, after all the PAHs were separated by reversed-phase liquid chromatography, analytes formed complexes with silver cation by mixing with silver nitrate solution. The complexes then transfer the molecular ion, [M] +, of the PAHs by charge transfer using in source collision-induced dissociation. The positive ion ESI mass spectra of all PAHs tested in this study showed [M] + as the base peak and abundant [M+Ag] +, [2M+Ag] + with very weak or no [2M+Ag] +. For the sample extraction, several solid-phase extraction parameters using the blue-chitin column were optimized. The limits of detection ( S/ N=3) of all PAHs for the spiked river water sample ranged from 0.001 to 0.03 ng/ml, and the detector responses were linear up to 1 ng/ml (correlation coefficients ≥0.0998). Repeatability and reproducibility were in the range from 4.3 to 6.8% and from 6.2 to 9.5%, respectively.

Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response

Electrospray ionization-mass spectrometry (ESI-MS) is a very promising tool for the analysis of phospholipid compositions, but is hampered by the fact that not all molecular species are detected with equal efficiency. We studied this and other issues that need to be taken into account to obtain truly quantitative compositional data. The key findings were as follows: First, the instrument response for both saturated and unsaturated phospholipid species decreased with increasing acyl chain length. This effect became increasingly prominent with increasing overall lipid concentration. Second, the degree of acyl chain unsaturation also had a significant effect on instrument response. At the highest concentration studied (10 pmol/microl), polyunsaturated species gave 40% higher intensity than the fully saturated ones. The effect of unsaturation diminished and nearly disappeared with progressive dilution. Third, the instrument response for the different head group classes varied markedly depending on the infusion solvent used. Notably, inclusion of ammonia in the infusion solvent eliminated sodium adduct formation in the positive ion mode, thus greatly simplifying the interpretation of the spectra. The fact that instrument response is dependent on many structural features, overall lipid concentration, solvent composition, and instrument settings makes it necessary to include several internal standards for each phospholipid class to obtain accurate data. Preferably, both unsaturated and saturated standards should be used. Finally, we quantified the major phospholipid classes of BHK cells using ESI-MS. The data agreed closely with those obtained with thin-layer chromatography and phosphorus analysis. This study indicates that quantitative compositional data can be obtained with ESI-MS, provided that proper attention is paid to experimental details, particularly the choice of internal standards.

Electrospray and atmospheric pressure chemical ionization tandem mass spectrometric behavior of eight anabolic steroid glucuronides

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most suitable ionization method to produce intense structure specific product ions and to examine the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG), androsterone (AG), 5α-estran-3α-ol-17-one (5α-NG), 5β-estran-3α-ol-17-one (5β-NG), 17α-methyl-5α-androstane-3α,17β-diol (5α-MTG), and 17α-methyl-5β-androstane-3α,17β-diol (5β-MTG), the last four being new compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity of the 4-ene-3-one system in the steroid structure favored the formation of protonated molecule [M + H] + in positive ion mode mass spectrometry (MS), whereas the steroid glucuronides with lower proton affinities were detected mainly as ammonium adducts [M + NH 4] +. The only ion produced in negative ion mode mass spectrometry was a very intense and stable deprotonated molecule [M − H] −. Positive ion ESI and APCI MS/MS spectra showed abundant and structure specific product ions [M + H − Glu] +, [M + H − Glu − H 2O] +, and [M + H − Glu − 2H 2O] + of protonated molecules and corresponding ions of the ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of the precursor ion provided distinction of 5α-NG and 5β-NG isomers and TG and ETG isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS spectra. It was shown that positive ion ESI MS/MS is the most promising method for further development of LC-MS methods for anabolic steroid glucuronides.

Fully automated determination of eserine N-oxide in human plasma using on-line solid-phase extraction with liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A sensitive and entirely automated solid-phase extraction/liquid chromatography/electrospray ionization tandem mass spectrometric (SPE/LC/ESI-MS/MS) method was developed and validated for the determination of eserine N-oxide (ENO), a cholinesterase inhibitor-like physostigmine in human plasma, for use in pharmacokinetic studies. ENO is light-sensitive and the use of a fully on-line process increased the reliability of the assay. Plasma samples previously mixed with neostigmine bromide to prevent in vitro degradation, and tacrine as internal standard (IS), were directly injected into the SPE/LC/ESI-MS/MS system. MS software piloted the overall system. MS/MS detection of ENO and the IS was performed in the positive ion ESI mode using multiple reaction monitoring. The linear calibration curve for ENO ranged from 25 pg ml(-1) to 12.5 ng ml(-1). The limit of quantitation was 25 pg ml(-1) with 250 microl of plasma injected. Precision, accuracy and stability tests were within the acceptable range and just one analyst is required to analyze 50 unknown samples a day five days per week, from the preparation of the samples (i.e. thawing and centrifugation) to data processing. A pilot pharmacokinetic study in three healthy volunteers treated with 4.5 mg of ENO (Génésérine3((R))) showed that the method was suitable for pharmacokinetic studies in humans.

Exploratory pharmacokinetics and brain distribution study of a neuropeptide FF antagonist by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry.

Dansyl-Pro-Gln-Arg-NH(2), an N-terminally modified tripeptide amide and a putative neuropeptide FF antagonist, was amenable to both positive-ion ESI and APCI. The protonated molecule yielded several fragment ions upon collision-induced dissociation in a quadrupole ion trap instrument for the development of LC/MS/MS assay methods. ESI clearly outperformed APCI in limits of detection, and was the method of choice for coupling with narrow-bore reversed-phase liquid chromatography to assess the pharmacokinetic profile and brain concentration of the neuropeptide FF antagonist in experimental animals. While plasma could be analyzed after rapid sample preparation, brain tissue required cleanup (solid phase extraction) and preconcentration before injection, and the assay was prone to matrix interference. This study indicated a rapid disappearance of dansyl-Pro-Gln-Arg-NH(2) from the plasma and the brain, and modest CNS bioavailability after intravenous administration to rats.