A.G. Brenton

Fingerprint profile of Ginkgo biloba nutritional supplements by LC/ESI-MS/MS

Ginkgo biloba is one of the most popular herb nutrition supplements, with terpene lactones and flavonoids being the two major active components. A fingerprint profile method was developed using a capillary HPLC/MS method which can identify more than 70 components from the G. biloba product. The method allows the flavonoids and terpene lactones to be detected simultaneously and information of both the parent ion and its fragmentation can be obtained in just one HPLC/MS run. Targeted post-acquisition analysis allows mass spectrometric information regarding the identification of flavonoid components to be easily distinguished from other data, however the same approach for terpene lactones was less successful due to dimer formation and requires further development. The fingerprint profiles of five commercial G. biloba nutritional supplements were obtained and compared; variation of some components among the samples was observed and fortification could be detected. In the quality control analysis of the G. biloba product this method could be viewed as complementary to specific quantitative analysis of some bioactive components of the herb.

MUC1 as a Discriminator between Endometrium from Fertile and Infertile Patients with PCOS and Endometriosis

CONTEXTnEndometrium of fertile women expresses progesterone-regulated Mucin 1 (MUC1) that carries selectin ligands recognized by the human blastocyst. Altered MUC1 expression at the time of implantation may contribute to endometrial infertility.nnnOBJECTIVEnThe aim was to assess the expression of MUC1 in the endometrium from polycystic ovary syndrome (PCOS), endometriosis, and fertile women in comparison with other hormone-regulated proteins [hydroxysteroid dehydrogenase (HSD) 1, HSD2, estrogen receptor (ER) and progesterone receptor (PR)].nnnDESIGN AND PATIENTSnEndometrial samples were obtained from 33 fertile patients, 26 ovulatory PCOS patients, 15 anovulatory PCOS patients, and 25 endometriosis patients.nnnMAIN OUTCOME MEASUREnImmunohistochemistry assessed the expression of MUC1 subunits ER, PR, HSD1, and HSD2 in endometrial epithelium. Endometrial MUC1 expression was quantified by immunoblots and RT-PCR. HSD1 and HSD2 expression was assayed by RT-PCR.nnnRESULTSnMUC1ND expression was significantly higher in ovulatory PCOS than in fertile and anovulatory PCOS patients, even after progesterone stimulation. MUC1ND and -CD expression was lower in anovulatory PCOS than in fertile patients. Only MUC1CD expression was lower in endometriosis patients. Endometrial ER expression was significantly higher in PCOS and endometriosis patients, whereas PR expression was significantly higher in PCOS than in fertile patients. The expression of HSD1 was significantly higher in anovulatory PCOS than in fertile patients. Expression of HSD2 was significantly higher in PCOS patients and lower in endometriosis patients.nnnCONCLUSIONnExpression of MUC1 subunits in the infertile endometrium is significantly different from fertile and appears to be a component of altered gene expression that potentially contributes to endometrial insufficiency.

Investigation of uremic analytes in hemodialysate and their structural elucidation from accurate mass maps generated by a multi-dimensional liquid chromatography/mass spectrometry approach

Historically, structural elucidation of unknown analytes by mass spectrometry alone has involved tandem mass spectrometry experiments using electron ionization. Most target molecules for bioanalysis in the metabolome are unsuitable for detection by this previous methodology. Recent publications have used high-resolution accurate mass analysis using an LTQ-Orbitrap with the more modern approach of electrospray ionization to identify new metabolites of known metabolic pathways. We have investigated the use of this methodology to build accurate mass fragmentation maps for the structural elucidation of unknown compounds. This has included the development and validation of a novel multi-dimensional LC/MS/MS methodology to identify known uremic analytes in a clinical hemodialysate sample. Good inter- and intra-day reproducibility of both chromatographic stages with a high degree of mass accuracy and precision was achieved with the multi-dimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) system. Fragmentation maps were generated most successfully using collision-induced dissociation (CID) as, unlike high-energy CID (HCD), ions formed by this technique could be fragmented further. Structural elucidation is more challenging for large analytes >270 Da and distinguishing between isomers where their initial fragmentation pattern is insufficiently different. For small molecules (<200 Da), where fragmentation data may be obtained without loss of signal intensity, complete structures can be proposed from just the accurate mass fragmentation data. This methodology has led to the discovery of a selection of known uremic analytes and two completely novel moieties with chemical structural assignments made.

Investigation of Solvent-Free MALDI-TOFMS Sample Preparation Methods for the Analysis of Organometallic and Coordination Compounds

An investigation of various solvent-free matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) sample preparation methods for the characterization of organometallic and coordination compounds is described. Such methods are desirable for insoluble materials, compounds that are only soluble in disadvantageous solvents, or complexes that dissociate in solution, all of which present a major difficulty to most mass spectrometry techniques. First-row transition metal acetylacetonate complexes, which have been characterized previously by solution preparation MALDI-TOFMS, were used to evaluate the various solvent-free procedures. These procedures comprise two distinct steps: the first being the efficient solids mixing (the mixing of sample and matrix), and the second being the effective transfer of the sample/matrix mixture to the MALDI target plate. This investigation shows that vortex mixing is the most efficient first step and that smearing using a microspatula is the most effective second step. In addition, the second step is shown to be much more critical than the first step in obtaining high-quality data. Case studies of truly insoluble materials highlight the importance of these techniques for the wider chemistry community.

Collisional excitation of CO: a study of the wigner spin rule

Abstract Vibrational excitation of CO by 3 keV H+, He+, N+, O+, Cl+ and Ar+ ions has been investigated by high resolution translational energy spectroscopy. The vibrational structures for the observed X1Σ+ → A1Π and X1Σ+ → a3Πr transitions of CO are clearly resolved, and the various reaction channels are unambiguously identified. The projectile ions are deliberately chosen for their distinctly different valence electronic configurations in a systematic study of the Winger spin conservation rule. In all ion-atom/molecule collisions involving simultaneous transitions in both partners, spin conservation dictates that a single-triplet (or doublet-quartet) transition in one partner necessitates observance of the following selection rule by the other collision partner: ΔS = 0, ± 1 with singlet-singlet transition forbidden. In every instance where CO molecule undergoes a X1Σ+ → a3Πr transition, this selection rule appears to be observed rigorously by the atomic ion.

Electronic excitation of atmospheric molecules by proton impact

Abstract Electronic and vibrational excitation of N 2 , O 2 , CO and NO by 3 keV protons has been investigated by means of high resolution translational energy spectroscopy. The dominant transitions ( X 1 Σ + g → a 1 Π g ) in N 2 and ( X 1 Σ + → A 1 Π) in CO are well resolved and the relative intensities for the vibrational excitations have been determined. The dominant structures in O 2 result from excitation to the B 3 Σ − u and A 3 Σ + u states which leads to dissociation of the O 2 molecule. The most prominent feature in NO is difficult to identify because of the existence of eleven doublet electronic states from the B ′ 2 Δ i (7.48 eV) to the S 2 Σ + (8.29 eV) state. Three well-defined peaks observed at 5.5 eV to 6.1 eV have been assigned to the transitions X 2 Π r ( v = 0) → A 2 Σ + ( v = 0, 1, 2) of NO.

Online immobilized metal affinity chromatography/mass spectrometric analysis of changes elicited by cCMP in the murine brain phosphoproteome

An automated online immobilized metal affinity chromatography/high-performance liquid chromatography mass spectrometric (IMAC-HPLC/MS/MS) method was developed to study cytidine 3,5-cyclic monophosphate (cCMP)-specific protein phosphorylation, analogous to a previously successful offline IMAC method using microvolume IMAC pipette tips. The optimized method identified murine brain phosphoproteins selectively modified by challenge with cCMP, using manual interpretation of the results to confirm both phosphorylation and selectivity of response to cCMP. A number of proteins identified by this strategy have potential roles in hyperproliferation, a previously reported response to elevated levels of cCMP.

The design and performance of a high resolution translational energy loss spectrometer

An instrumental development is described that achieved high energy resolving power when studying collisions between fast ion beams, at keV energies, interacting with thermal molecular and atomic gaseous targets. This technique is referred to as translational energy spectroscopy (TES) and can reveal spectroscopic information on the states of the participating species, the dynamics of the collision, lifetimes of species formed, state-to-state collision cross-sections and populations of states. The instruments described are based on an unique symmetrical arrangement of cylindrical electrostatic analysers. These are placed in a double focusing ion-optical geometry leading to very small first and second order image aberrations, sufficient to obtain a primary energy resolving power of 105. Two such instruments were developed and their relative performances are compared experimentally and through computer modelling; the second, and larger, instrument has an improved sensitivity by an order of magnitude and a three times increase in resolving power. This has enabled collision spectroscopy to achieve performance similar to electron energy-loss spectroscopy and photoelectron spectroscopy, with a resolution on the unscattered ion beam measured to be 0.01 eV, at 3 keV collision energy, on energy loss processes of ≥u20090.03 eV. These developments allow TES to observe, (i) the vibrational/electronic spectroscopy of keV ion/molecule collisions; (ii) spin-selective spectra; (iii) the highly excited states close to and beyond the ionization limit of the target species; and (iv) involatile targets using a high temperature cell. The validity of optical quantum selection rules, relying on the electric-dipole interaction governing photon-induced transitions, has been tested for TES, which relies on the more general ion/neutral interaction potential responsible for collisional excitation. Spin conservation was found to be strongly adhered to, whilst orbital angular momentum and total angular momentum were not. The specific selection rules for homonuclear diatomics g g and u u were found to be allowed in TES, in contradiction to the case of optical spectroscopy, whilst there was some preliminary evidence that the optical Σ+–Σ− symmetry-forbidden transition was also forbidden for the TES reactions studied. For H2+ projectiles, excitation to triplet states is strongly favoured over excitation to singlet states.

Collisional excitation of the Meinel and first negative systems of N+2

Abstract High-resolution translational energy spectroscopy has been performed of N+2 ions scattered off Ar, N2, O2 and NO. Intense excitations and de-excitations of the Meinel system of N+2 (A2Πu ← X2∑+2) were observed in all collision gases. Transitions in the first negative system N+2 (B3∑+u ← X2g) were also detected, and well-resolved in the case with Ar as collision gas. Simultaneous transitions involving excitation of the Meinel system and the a1Δg state of O2 were observed with O2 as collision gas.

Spin conservation in collision of C+ ions with atomic and molecular targets

Abstract High resolution translation energy spectroscopy (TES) has been performed on C+ ions in collision with Ar, CO, O2 and NO. The target species included atoms and molecules in singlet, triplet and doublet ground states. The observed reaction channels were discussed in the light of a simple selection rule based on the Wigner spin conservation requirements. All the identified channels were found to be in accord with these requirements. The fractional population of the 4P state in the C+ ion beam has been estimated from the relative intensities for the C+ (2P ↔ 4P) transitions obtained from the TES spectrum of C+ on O2. The fraction of C+ (4P) was found to be about 10% of ions produced from CH4 precursor gas at a nominal electron ionizing energy of 100 eV.