Determination of estrogens and progestogens by mass spectrometric techniques (GC/MS, LC/MS and LC/MS/MS)

Steroid sex hormones and related synthetic compounds have been shown to provoke alarming estrogenic effects in aquatic organisms, such as feminization, at very low concentrations (ng/L or pg/L). In this work, different chromatographic techniques, namely, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), are discussed for the analysis of estrogens, both free and conjugated, and progestogens, and the sensitivities achieved with the various techniques are inter-compared. GC/MS analyses are usually carried out after derivatization of the analytes with bis(trimethylsilyl)trifluoroacetamide (BSTFA). For LC/MS and LC/MS/MS analyses, different instruments, ionization techniques (electrospray (ESI) and atmospheric pressure chemical ionization (APCI)), ionization modes (negative ion (NI) and positive ion (PI)) and monitoring modes (selected ion monitoring (SIM) and selected reaction monitoring (SRM)) are generally employed. Based on sensitivity and selectivity, LC/ESI-MS/MS is generally the method of choice for determination of estrogens in the NI mode and of progestogens in the PI mode (instrumental detection limits (IDLs) 0.1-10 ng/mL). IDLs achieved by LC/ESI-MS in the SIM mode and by LC/ESI-MS/MS in the SRM mode were, in general, comparable, although the selectivity of the latter is significantly higher and essential to avoid false positive determinations in the analysis of real samples. Conclusions and future perspectives are outlined.     

A new technique (COMSPARI) to facilitate the identification of minor compounds in complex mixtures by GC/MS and LC/MS: tools for the visualization of matched datasets

In the rapidly growing field of metabolomics, it is common to analyze complex biological samples by chromatography coupled to mass spectrometry. While several techniques are available for the detection of significant peaks in individual samples, it is still difficult to determine small differences between similar samples. Using conventional software, visual inspections of individual chromatograms or individual mass spectra are often of little use because the differences in the composition of small molecules are too small to be recognizable. Thus, we developed a new approach to visualizing mass spectral datasets using a tool that allows one to easily detect these small differences between mass spectra and chromatograms derived from matched samples. Using these tools on extracts from wild-type and methyltransferase knockout strains of the yeast Saccharomyces cerevisiae, we were able to readily identify those mass spectra in our data sets that were different between the wild-type and the knockout extracts and to identify the molecules involved. The software was also successfully applied to a set of LC/MS data from peptide digests that were performed with identical substrates but different enzymes. We have named this visualization tool COMSPARI (COMparision of SPectrAl Retention Information) and are making the software publicly available via Internet at.     

Identification of the Metabolite of 7-(4-Chlorbenzyl)-7,8,13,13a-tetrahydroberberine chloride in Rat Bile by LC/MS and LC/NMR

7-(4-ChiorbenZyl)-7,8,l3,13a-tetrahydroberbenne(CTHB),anewchemicalenhty,swteslzedinResearchDivisionofMedicinalChemistryofChinaPharmaceuhcalUniversity(CPU),hasshownitseffectsagatristarrhtwainstudiesconductedinCPU.TheseinveshgahonssuggestthatCTasispromisingtobedevelopedasanewanharrhythmicagent.EXPERIMEwrAL:ChenucalshiftsofallprotonsofCTHBwereassignedby'H-'HCoSYand"C-'HCoSYBilewasobtsinedfromSDratSallercannulahonofthebilinyductandwascollectedinice-cooledtUbesallerintravenousadr…     

Equilibrium and structure of the Al(III)-ethylenediamine-N,N'-bis(3-hydroxy-2-propionate) (EDBHP) complex. A multi-method study by potentiometry, NMR, ESI MS and X-ray diffraction

The equilibrium and structure of the complex formed by Al(III) and ethylenediamine-N,N'-bis(3-hydroxy-2-propionate) (EDBHP2-) have been studied using pH-potentiometry, 1H and 27Al NMR, ESI MS and single crystal X-ray diffraction methods. The EDBHP ligand is a strong Al-binder in aqueous solution for pH between 4 and 8 and for c(Al) = c(EDBHP)> or = 0.1 mmol dm(-3). The dominating complex identified by ESI MS and potentiometry is a neutral dimer, Al2L2(OH)2, with logbeta(22-2) = 14.16 +/- 0.03. In the solid Al2(EDBHP)2(OH)2.2H2O the Al(III) ions are connected through a double hydroxo bridge. Both four-dentate organic ligands are coordinated terminally through two carboxylate groups and two N-donors forming three five-membered chelate rings. The hydroxyl groups of the ligand EDBHP remain protonated and are not coordinated to the aluminium ions. The structure and composition of the dimer are very likely the same in solution and the solid state.     

Maximum-quantum (MaxQ) NMR for the speciation of mixtures of phenolic molecules

The spectroscopic determination of phenolic molecules by means of multiple-quantum (MQ) NMR is demonstrated. Several classes of molecules (simple phenols, flavonols, secoiridoids and lignans) were unambiguously characterized in one pot analysis in an extract of extra virgin olive oil (EVOO).     

Simultaneous determination of VD2, VD3, 25(OH) D2, and 25(OH) D3 in human plasma using electrospray LC–MS/MS as well as its application to evaluate VD plasma levels in depressive,schizophrenic patients and healthy individuals

Vitamin D measurements in biological fluids by liquid chromatography–tandem mass spectrometry (LC–MS/MS) have been widely used but remain challenging at very low concentration levels. Rapid, high recovery, sensitive and reliable measurements of vitamin D, as well as its primary metabolites using LC–MS/MS are urgently needed for a routine clinical laboratory. Herein, we reported a novel electrospray LC–MS/MS method for determining vitamin D and its primary metabolites using the supported liquid extraction method to achieve higher recoveries, with optimized pH values to achieve optimal derivatization efficiency for higher sensitivity and selected chromatographic conditions to shorten the separation time. The method has been validated with respect to selectivity, recovery, matrix effects, accuracy and precision, stabilities, carryover and dilution effects. The method has been successfully applied to quantify the VD plasma concentrations of depressive, schizophrenic patients and healthy individuals. The result showed that there were significant differences in plasma VD levels between mental disorder patients with healthy individuals, and the total VD levels in mental disorder patients were much higher than healthy individuals, which might require larger clinical samples for validation.     

Application of a flow-tunable, serially coupled gas chromatographic capillary column system for the analysis of complex mixtures

Summary A capillary column gas chromatographic system employing two serially coupled fused-silica columns and a simple coupling element is described. The system is operated in flow-tunable mode (flow-tunable tandem system). The very fact of continuous tuning over a large polarity (selectivity) range, ultimately determined by the two constituent columns, offers several possibilities in the analysis of complex mixtures. In this paper two applications are discussed in detail: optimization of peak separation and peak identification. For these applications it is feasible to use, retention data collected from experiments on the tandem system, and empirical formulas. A relatively simple theoretical mathematical model valid for the flow-tunable tandem system, however, furnishes an easy way of calculating retention data on the system from data collected from the individual single columns, thus, creating a new possibility for optimization and peak identification. Optimization and peak identification processes using the empirical and theoretical models are both demonstrated by analysis of solvent mixtures. Dedicated to the memory of Dr. Tibor Tóth Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

The formation of the complex pentacyano(3-pyrazincarboxylate)ferrate(II) in various water-cosolvent mixtures

This article presents the kinetic of formation of pentacyano(3-pyrazincarboxylate)ferrate(II) from pyrazincarboxylate and pentacyanoaquoferrate(II) ions in various isodielectric water-cosolvent mixtures at 298 K. The rate law is in the form d[Fe(CN)₅(3-pzCO₂)^4?]/dt = ??_f[Fe(CN)₅H₂O^?3] [pzCO₂^?]. Plots of log(??_f/dm³ mol^?1 s^?1) vs. D (where D_m is the bulk dielectric constant of the medium) and log(??_f/dm³ mol^?1 s^?1) vs. the Grunwald-Winstein parameter are nonlinear for some of the mixtures and to each mixture corresponds a different behavior in respect to the above parameters. The plots of log(??_f/dm³ mol^?1 s^?1) vs. the mol fraction of water are straight lines over the entire composition range studied, except for the water-methanol mixture. It is evident that the solvation phenomenon plays a dominant role and that the rate of formation is mediated by the dual solvent vectors, the overall basicity and acidity of the solvent mixtures.     

Development of an LC/MS method for the trace analysis of triacetone triperoxide (TATP)

The detection and quantification of triacetone triperoxide (TATP) using LC/MS is investigated. GC/MS analysis of TATP is hindered by stationary phase activation in very short periods of time. Due to the lower temperatures used in LC. this problem is not encountered. This study presents a method that is suitable for the detection of TATP at levels as low as 100 pg microl(-1) (10 ng per 100 microl). Initial findings are also reported for the investigation of a secondary chromatographic peak, which is thought to be caused by separation of two conformers. This study concludes that LC/MS is a suitable technique for the analysis of trace levels of TATP.     

DNA recognition by the anthracycline antibiotic respinomycin D: NMR structure of the intercalation complex with d(AGACGTCT)2

Respinomycin D is a member of the anthracycline family of antitumour antibiotics that interact with double stranded DNA through intercalation. The clinical agents daunomycin and doxorubicin are the most well-studied of this class but have a relatively simple molecular architecture in which the pendant daunosamine sugar resides in the DNA minor groove. Respinomycin D, which belongs to the nogalamycin group of anthracyclines, possesses additional sugar residues at either end of the aglycone chromophore that modulate the biological activity but whose role in molecular recognition is unknown. We report the NMR structure of the respinomycin D-d(AGACGTCT)2 complex in solution derived from NOE restraints and molecular dynamics simulations. We show that the drug threads through the DNA double helix forming stabilising interactions in both the major and minor groove, the latter through a different binding geometry to that previously reported. The bicycloaminoglucose sugar resides in the major groove and makes specific contacts with guanine at the 5'-CpG intercalation site, however, the disaccharide attached at the C4 position plays little part in drug binding and DNA recognition and is largely solvent exposed.     

A program for the processing of analytical data (DPP)

Computer-assisted interactive data presentation and analysis facilities are needed to handle the vast amount of information produced by automated instruments. The data processing program, DPP, presented here in a FORTRAN-77 program designed to solve this problem. The program is equipped with a leading verb command language for input and job scheduling, thus providing an efficient and user-friendly operator/program interface, and with a data-base organization that accommodates a wide variety of data structures. Data presentation and analysis procedures include tabulation and plotting, regression analysis, non-linear least-squares fitting, polynomial fitting, principal component analysis, hierarchal clustering and non-hierarchal clustering. Data matrices with up to 10 000 data points, distributed over a maximum of 3000 variables and 3000 samples, can be examined. Because of the open-ended structure of the program, it is straightforward to incorporate additional data analysis procedures when they are needed. Recent applications are discussed.     

Synthesis,characterization and catalytic activity of Rh (PPh3)2 (en)Cl complex

Ethylenediamine bis(triphenylphosphine) monochlororhodium has been prepared by the interaction of Wilkinson's catalyst and ethylenediamine in benzene. The complex has been isolated and characterized by conventional and spectroscopic methods. The catalytic activity of the complex was investigated for the hydrogenation of 1-octene as a model reaction at a hydrogen pressure of 1 atmosphere (101 kPa) using methanol as a solvent. The influence of various factors such as catalyst, substrate concentrations and temperature have been studied. The hydrido complex has been identified as an intermediate product by IR and NMR studies. The experimental data are in accordance with a rate expression of the form: .     

A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25‐hydroxyvitamin D₃ [25(OH)D₃, the best‐established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI‐MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels–Alder reaction with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione followed by acetylation, to enhance the detectability of 25(OH)D₃ in ESI‐MS/MS and to separate 25(OH)D₃ from 3‐epi‐25‐hydroxyvitamin D₃ [3‐epi‐25(OH)D₃], a potent interfering metabolite. 25(OH)D₃ was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 μL of whole blood), purified using an Oasis HLB^® cartridge, and subjected to derivatization prior to analysis with LC/ESI‐MS/MS. 25‐Hydroxyvitamin D₄ was used as the internal standard. This method was reproducible (intra‐ and inter‐assay RSDs, <6.9%) and accurate (analytical recovery, 95.2–102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D₃ in neonatal DBSs and detection of vitamin D deficiency without interference from 3‐epi‐25(OH)D₃.     

Theoretical characterization of the gas-phase O(3)HO hydrogen-bonded complex.

We report a theoretical study on two gas-phase hydrogen-bonded complexes formed between ozone and hydroxyl radical that have relevance to atmospheric chemistry. This study was carried out by using CASSCF, CASPT2, QCISD, and CCSD(T) theoretical approaches in conjunction with the 6-311+G(2df,2p) and aug-cc-pVTZ basis sets. Both complexes have a planar structure and differ from each other in the orientation of the electronic density of the unpaired electron associated with the HO radical moiety. Our calculations predict their stabilities to be 0.87 and 0.67 kcal mol(-1), respectively, at 0 K and show the importance of anharmonic effects in computing the red shift of the HO stretch originating from the hydrogen-bonding interaction. We also report two transition states involving the movement of the HO moiety on the potential energy surfaces of these hydrogen-bonded complexes.     

Compehensive two-dimensional gas chromatography (GC×GC) and its applicability to the characterization of complex (petrochemical) mixtures

In general, petrochemical products contain only a limited number of chemical classes of compounds (sample dimensionality). The enormous number of individual components within these classes, however, soon puts limitations upon a single chromatographic technique when it comes to adequate characterization of these products. Comprehensive two-dimensional gas chromatography (GC×GC) clearly opens the possibility of estimating the composition of hydrocarbon mixtures in a far more detailed fashion than hitherto possible. Although the emphasis of papers of GCxGC thus far almost exclusively applies to the unsurpassed peak-capacity, in the oil industry there is a need for characterization, rather than for analyzing all the individual compounds. In principle a GCxGC system can provide an almost perfect match between its intrinsic properties and the dimensionality of oil samples. To establish the applicability of GCxGC towards petrochemical analytical challenges, a commercially aavailable prototype instrument was subjected to an exhaustive characterization of a typical hydrocarbon precess stream and a fast characterization of a light gas oil. Although there are no fundamental limitations towards the quantitative aspects of a GCxGC system, this paper confines itself to qualitative results only. Quantitative aspects of GCxGC will be published in a forthcoming paper.     

Determination of perfluorooctanoic acid (PFOA) extractable from the surface of commercial cookware under simulated cooking conditions by LC/MS/MS

Salts of pentadecafluorooctanoic acid (PFOA) are polymerization aids used in the manufacture of fluoropolymers; one of the applications of fluoropolymers is the coating of metal cookware products. A method was developed to determine if PFOA might be present in and extracted from the surface of commercial frying pans coated with a DuPont fluoropolymer under simulated cooking conditions. Commercial grade cookware was obtained, then extracted with water and ethanol/water mixtures at 100 and 125 degrees C, and the resulting extracts were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). Detection and quantification limits as low as 100 pg cm(-2) were demonstrated. None of the fluoropolymer treated cookware samples analyzed showed detectable levels of PFOA when extracted under simulated cooking conditions.     

A hydrogen-1, nitrogen-15, and chlorine-35 NMR coordination study of Lu(ClO4)3 and Lu(NO3)3 in aqueous solvent mixtures

A coordination study of Lu(III) has been carried out for the nitrate and perchlorate salts in aqueous mixtures of acetone-d₆ and Freon-12 by¹H,¹⁵N and³⁵Cl NMR spectroscopy. At temperatures lower than –90°C, proton and ligand exchange are slow enough to permit the direct observation of¹H resonance signals for coordinated and free water molecules, leading to an accurate measure of the Lu(III) hydration number. In perchlorate solution, in the absence of inner-shell ion-pairing, Lu(III) exhibits a maximum coordination number of six over the allowable concentration range of study, contrasting markedly with the report of values of six to nine or greater as determined by a similar NMR method. The absence of contact ion-pairing was confirmed by³⁵Cl NMR chemical shift and linewidth measurements. Extensive ion-pairing was observed in the nitrate solutions as reflected by the lower Lu(III) hydration numbers of two to three in these systems, the observation of two coordinated water signals, and¹⁵N NMR signals for two complexes. The¹H and¹⁵N NMR spectra and the hydration number could be accounted for by the presence of (H₂O)₄Lu(NO₃)²⁺ and (H₂O)₂Lu(NO₃) ₂ ¹⁺ .