Characterization of gas-phase molecular interactions on differential mobility ion behavior utilizing an electrospray ionization-differential mobility-mass spectrometer system

Differential mobility spectrometry (DMS) is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal/noise ratios, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and potential for rapid analyte quantitation. The introduction of a new ESI-DMS-MS system and its utilization to aid in the understanding of DMS separation theory is described. A current contribution to DMS separation theory is one of an association/dissociation process between ions/molecules in the gas phase during the differential mobility separation. A model study was designed to investigate the molecular dynamics and chemical factors influencing the theorized association/dissociation process, and the mechanisms by which these gas-phase interactions affect an ion's DM behavior. Five piperidine analogues were selected as model analytes, and three alcohol drift gas dopants/modifiers were used to interrogate the analyte ions in the gas phase. Two proposed DMS separation mechanisms, introduced as Core and Fa?ade, corresponding to strong and weak attractions between ions/molecules in the gas phase, are detailed. The proposed mechanisms provide explanation for the observed changes in analyte separation by the various drift gas modifiers. Molecular modeling of the proposed mechanisms provides supportive data and demonstrates the potential for predictive optimization of analyte separation based on drift gas modifier effects.     

On-line capillary electrophoresis/microelectrospray ionization-tandem mass spectrometry using an ion trap storage/time-of-flight mass spectrometer with SWIFT technology.

The development of a system capable of the speed required for on-line capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) of tryptic digests is described. The ion trap storage/reflectron time-of-flight (IT/reTOF) mass spectrometer is used as a nonscanning detector for rapid CE separation, where the peptides are ionized on-line using electrospray ionization (ESI). The ESI produced ions are stored in the ion trap and dc pulse injected into the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology generated by software and hardware developed in our lab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion isolation and TICKLE activation/fragmentation voltage waveforms to generate MS/MS at a rate as high as 10 Hz so that the MS/MS spectra can be optimized on even a 1-2 s eluting peak. In CE separations performed on tryptic digests of dogfish myelin basic protein (MBP) where eluting peaks 4-8 s wide are observed, it is demonstrated that an acquisition rate of 4 Hz provides > 20 spectra/peak and is more than sufficient to provide optimized MS/MS spectra of each of the eluting peaks in the electropherogram. The detailed structural analysis of dogfish MBP including several posttranslational modifications using CE-MS and CE-MS/MS is demonstrated using this method with < 10 fmol of material consumed.     

Choosing between atmospheric pressure chemical ionization and electrospray ionization interfaces for the HPLC/MS analysis of pesticides

An evaluation of over 75 pesticides by high-performance liquid chromatography/mass spectrometry (HPLC/MS) clearly shows that different classes of pesticides are more sensitive using either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). For example, neutral and basic pesticides (phenylureas, triazines) are more sensitive using APCI (especially positive ion). While cationic and anionic herbicides (bipyridylium ions, sulfonic acids) are more sensitive using ESI (especially negative ion). These data are expressed graphically in a figure called an ionization-continuum diagram, which shows that protonation in the gas phase (proton affinity) and polarity in solution, expressed as proton addition or subtraction (pKa), is useful in selecting APCI or ESI. Furthermore, sodium adduct formation commonly occurs using positive ion ESI but not using positive ion APCI, which reflects the different mechanisms of ionization and strengthens the usefulness of the ionization-continuum diagram. The data also show that the concept of "wrong-way around" ESI (the sensitivity of acidic pesticides in an acidic mobile phase) is a useful modification of simple PKa theory for mobile-phase selection. Finally, this finding is used to enhance the chromatographic separation of oxanilic and sulfonic acid herbicides while maintaining good sensitivity in LC/MS using ESI negative.     

Structural characterization of chemically derivatized oligosaccharides by nanoflow electrospray ionization mass spectrometry.

Oligosaccharides released from several glycoproteins were derivatized with either 4-aminobenzoic acid 2-(diethylamino)ethyl ester (ABDEAE) (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) or 2-aminopyridine. The resulting derivatives were analyzed on a nanoflow electrospray ionization (ESI) quadrupole-inlet time-of-flight mass spectrometer using the low-energy collision-induced dissociation technique. In the MS/MS spectra, the oxonium (b or internal series) and y series ions, which are derived from the multiply charged precursor ions, were predominant and were used for the structural readout. Some oxonium ions that were observed in the low-mass region, but that were not found in the PSD analyses (Mo, W.; et al. Anal. Chem. 1998, 70, 4520-4526), rendered a more detailed structural insight. The oxonium ions at m/z 512.2, which are derived from the fucosylated oligosaccharides of immunoglobulin Y and thyroglobulin, were observed, suggesting that fucosylation had occurred proximal to the outer nonreducing terminus. In addition, the data herein show that structural elucidation can be routinely achieved at a low sample concentration. For the case of ABDEAE derivatives, this can be achieved at the 50 fmol/microL level and with the actual sample consumption at the attomole level using nanoflow ESI MS/MS.     

Evaluating the separation of amphetamines by electrospray ionization ion mobility spectrometry/MS and charge competition within the ESI process.

The rapid increase in amphetamine abuse for recreational purposes has created a need for fast analysis and detection methodologies. For the first time, we show the separation of six amphetamines by ESI-IMS/MS. A complete analysis can be performed in 70 s, which is faster than traditional chromatographic techniques. In addition, ESI-IMS/MS was found to provide low detection limits for the six compounds (15.4 ppb for ethylamphetamine). Charge competition between amphetamines was found to occur at high amphetamine concentrations. The degree of preferential ionization was dependent on the functional group placed on the amine. Both one-analyte and two-analyte calibration curves were evaluated on the basis of the ion evaporation model. Evaporation rates were determined for the six amphetamines, and the rates were correlated with the degree of selective ionization. Evaluation of three typical ESI solvent compositions showed that the addition of a modifier (acetic acid and formic acid) enhanced the degree of preferential ionization for some amphetamines and increased the effect of charge competition. The solvent studies show the complexity of ESI and provide possible strategies for altering the amount of charge competition between analytes. Overall, ESI-IMS/MS appears to be a promising technique because of its sensitivity and rapid separation times for the amphetamines in aqueous samples; however, further research employing biological samples is required before it can be recommended as a mainstream technique.     

Gradient tandem mass spectrometry interfaced with ion mobility separation for the characterization of supramolecular architectures

Traveling wave ion mobility mass spectrometry (TWIM MS) was combined with gradient tandem mass spectrometry (gMS(2)) to deconvolute and characterize superimposed ions with different charges and shapes formed by electrospray ionization (ESI) of self-assembled, hexameric metallomacrocycles composed of terpyridine-based ligands and Cd(II) ions. ESI conditions were optimized to obtain intact hexameric cation assemblies in a low charge state (2+), in order to minimize overlapping fragments of the same mass-to-charge ratio. With TWIM MS, intact hexameric ions could be separated from remaining fragments and aggregates. Collisional activation of these hexameric ions at varying collision energies (gMS(2)), followed by TWIM separation, was then performed to resolve macrocyclic from linear hexameric species. Because of the different stabilities of these architectures, gMS(2) changes their relative amounts, which can be monitored individually after subsequent ion mobility separation. On the basis of this unique strategy, hexameric cyclic and linear isomers have been successfully resolved and identified. Complementary structural information was gained by the gMS(2) fragmentation pattern of the metallosupramolecules, acquired by collisionally activated dissociation after TWIM dispersion. TWIM MS interfaced with gMS(2) should be particularly valuable for the characterization of a variety of supramolecular polymers, which often contain isomeric architectures that yield overlapping fragments and aggregates upon ESI MS analysis.     

Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/ QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/ MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.     

Ionization and fragmentation of humic substances in electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) was combined with ultra-high-resolution Fourier transform-ion cyclotron resonance mass spectrometry (FTICR MS) to characterize complex humic and fulvic acid mixtures. Lower than expected molecular weight distributions previously observed for humics when analyzed by ESI-MS have fueled speculation about a bias in favor of low molecular weight. Multiply charged ions, ionization suppression, and sample fragmentation have all been suggested as sources of this low molecular weight bias. In this work, resolution of the individual components of humic mixtures within a 1 mass-to-charge unit window was accomplished by FTICR MS at 9.4 T. At mass resolving powers between 60,000 (high mass) and 120,000 (low mass), it was possible to determine that virtually all ions present in spectra of Suwannee River fulvic and humic acid are singly charged, thus eliminating inadequate accounting for multiply charged ions as a primary source of any low molecular weight bias. The high-resolution mass spectra also revealed the presence of molecular families containing ions that differ from each other in degree of saturation, functional group substitution (primarily CH vs N and CH4 vs O), and number of CH2 groups. Ionization suppression and ion fragmentation were addressed for humic and fulvic acid mixtures and well-characterized poly(ethylene glycol) (PEG) mixtures with average molecular weights of 8000 and 10,000. Although these high molecular weight PEG mixtures fragment extensively under traditional positive-ion mode ESI conditions, similar fragmentation could not be confirmed for humic and fulvic acid mixtures.     

Rapid separation and quantitative analysis of peptides using a new nanoelectrospray- differential mobility spectrometer-mass spectrometer system

Differential mobility spectrometry (DMS) (see Buryakov, I. A.; Krylov, E. V.; Nazarov, E. G.; Rasulev, U. Kh. Int. J. Mass Spectrom. Ion Processes 1993, 128, 143-148), also commonly referred to as high-field asymmetric waveform ion mobility spectrometry (FAIMS) (see Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszcyk, M. S. Rev. Sci. Instrum. 1998, 69, 4094-4105), is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal-to-noise, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and the potential for rapid analyte quantitation. In this report, we investigate the use of our nanoESI-DMS-MS system to demonstrate differential mobility separation of peptides. The formation of higher order peptide aggregate ions (ion complexes) via electrospray ionization and the negative impact this has on DMS peptide separation are examined. The successful use of differential mobility drift gas modifiers (dopants) to reduce aggregate ion size and improve DMS peptide ion separation is presented. Following optimization of DMS peptide separation conditions, we examined next the feasibility of a new analytical platform which uses direct sample infusion with nanoESI-DMS-MS for ultrarapid analyte quantitation. Quantitation of a selected peptide from a semicomplex peptide mixture is presented. Initial feasibility results with this new approach demonstrate good accuracy and reproducibility, as well as an absolute mass sensitivity of 6.8 amol and a minimum dynamic range of 2500 for the peptide of interest. This report offers a first look at utilizing nanoESI-DMS-MS to create an ultrarapid (under 5 s) quantitative analysis platform and its potential in the high-throughput arena. Each ion separation technique, DMS and MS, offers orthogonal ion separation to one another, enhancing the overall specificity for this quantitative approach.     

Microfabricated differential mobility spectrometry with pyrolysis gas chromatography for chemical characterization of bacteria

A microfabricated drift tube for differential mobility spectrometry (DMS) was used with pyrolysis-gas chromatography (py-GC) to chemically characterize bacteria through three-dimensional plots of ion intensity, compensation voltage from differential mobility spectra, and chromatographic retention time. The DMS analyzer provided chemical information for positive and negative ions simultaneously from chemical reactions between pyrolysis products in the GC effluent and reactant ions of H+(H2O)n and O2-(H2O)n in air at ambient pressure. Authentic standards for chemicals formed in the pyrolysis of bacteria showed favorable matches with plots from py-GC/DMS analysis and were supported by py-GC/MS results. These and other yet-unidentified constituents provided a means to distinguish Escherichia coli from Micrococcus luteus. A Gram-positive spore former (Bacillus megaterium) was distinguished by an abundant peak for crotonic acid evident in positive and negative ions and not observed with M. luteus. In contrast, plots from py-GC/DMS of lipid A and lipoteichoic acid showed poor matches to plots for a Gram-negative (E. coli) bacterium and a Gram-positive (M. luteus) bacterium and the differences were attributed to differences in genus sources of the biopolymers. A significant percentage of the chemical information available in py-GC/DMS is unidentified, and the analytical utility must be established. Precision in the chemical measurement was determined as +/- 0.2 V, 10% relative standard deviation (RSD), and +/- 0.05 min for compensation voltage, peak intensity, and retention time, respectively. The minimum number of total bacteria (cell forming units) detected was 6000 though detection limits and resolution could be varied by the magnitude of the separation voltage in the differential mobility spectrometer.     

Microchip electrospray: improvements in spray and signal stability during gradient elution by an inverted postcolumn makeup flow

Dynamic changes in mobile phase composition during high-performance liquid chromatography (HPLC) gradient elution coupled to mass spectrometry (MS) sensitively affect electrospray modes. We investigate the impact of the eluent composition on spray stability and MS response by infusion and injection experiments with a small tetrapeptide in water-acetonitrile mixtures. The employed HPLC/electrospray (ESI)-MS configuration uses a microchip equipped with an enrichment column, a separation column, and a makeup flow (MUF) channel. One nano pump is connected to the separation column, while a second one delivers solvent of exactly inverted composition to the MUF channel. Both solvent streams are united behind the separation column, before the ESI tip, such that the resulting electrosprayed solution always has identical composition during a gradient elution. Analyte peak parameters without and with MUF compensation are determined and discussed with respect to the electrospray mode and eluent composition. The postcolumn MUF significantly improves spray and signal stability over the entire solvent gradient, without compromising the performance of the HPLC separation column. It can also be conveniently implemented on microchip platforms.     

Exact masses and chemical formulas of individual Suwannee River fulvic acids from ultrahigh resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectra

Molecular formulas have been assigned for 4626 individual Suwannee River fulvic acids based on accurate mass measurements from ions generated by electrospray ionization and observed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Formula assignments were possible because of the mass accuracy of FTICR MS at high field (9.4 T) and the regular mass spacing patterns found in fulvic acid mixtures. Sorting the 4626 individually observed ions according to Kendrick mass defect and nominal mass series (z* score) revealed that all could be assigned to 1 of 266 distinct homologous series that differ in oxygen content and double bond equivalence. Tandem mass spectrometry based on infrared multiphoton dissociation identified labile fragments of fulvic acid molecules, whose chemical formulas led to plausible structures consistent with degraded lignin as a source of Suwannee River fulvic acids.     

Estimates of protein surface areas in solution by electrospray ionization mass spectrometry

The extent of multiple charging of protein ions in electrospray ionization (ESI) mass spectra depends on the solvent-exposed surface area, but it may also be influenced by a variety of other extrinsic and intrinsic factors. Gas-phase ion chemistry (charge-transfer and charge-partitioning reactions) appears to be the major extrinsic factor influencing the extent of protonation as detected by ESI MS. In this work, we demonstrate that under carefully controlled conditions, which limit the occurrence of the charge-transfer reactions in the gas phase, charge-state distributions of protein ions can be used to assess the solvent-exposed surface area in solution. A set of proteins ranging from 5-kDa insulin to 500-kDa ferritin shows a clear correlation between the average charge in ESI mass spectra acquired under native conditions and their surface areas calculated based on the available crystal structures. An increase of the extent of charge-transfer reactions in the ESI interface results in a noticeable decrease of the average charge of protein ions across the entire range of tested proteins, while the charge-surface correlation is maintained. On the other hand, the intrinsic factors (e.g., a limited number of basic residues) do not appear to play a significant role in determining the protein ion charge. Based on these results, it is now possible to obtain estimates of the surface areas of proteins and protein complexes, for which crystal structures are not available. We also demonstrate how the ESI MS measurements can be used to characterize protein-protein interaction in solution by providing quantitative information on the subunit interfaces formed in protein associations.     

High-resolution field asymmetric waveform ion mobility spectrometry using new planar geometry analyzers

Field asymmetric waveform ion mobility spectrometry (FAIMS) has emerged as a powerful tool of broad utility for separation and characterization of gas-phase ions, especially in conjunction with mass spectrometry (MS). In FAIMS, ions are filtered by the dependence of mobility on electric field while being carried by gas flow through the analytical gap between two electrodes of either planar (p-) or cylindrical (c-) geometry. Most FAIMS/MS systems employ c-FAIMS because of its ease of coupling to MS, yet the merits of the two geometries have not been compared in detail. Here, a priori simulations reveal that reducing the FAIMS curvature always improves resolution at equal sensitivity. In particular, the resolving power of p-FAIMS exceeds that of c-FAIMS, typically by a factor of 2-4 depending on the ion species and carrier gas. We have constructed a new planar FAIMS incorporating a curtain plate interface for effective operation with an ESI ion source and joined to an MS using an ion funnel interface with a novel slit aperture. The resolution increases up to 4-fold over existing c-FAIMS, even though the analysis is approximately 2 times faster. This allows separation of species not feasible in previous FAIMS studies, e.g., protonated leucine and isoleucine or new bradykinin isomers. The improvement for protein conformers (of ubiquitin) is less significant, possibly because of multiple unresolved geometries.     

Designing polymeric hydrogen-bonded networks using 4,4-dipyridyl and aromatic carboxylic acids containing N–H substituents

Three adduct structures of 4,4-dipyridyl (4,4-DP) have been prepared and studied using single-crystal X-ray techniques. These are indole-2-carboxylic acid [(4,4-DP)(ICA)₂] 1, 3-aminobenzoic acid [(4,4-DP)(3-ABA)] 2, and 4-aminobenzoic acid [(4,4-DP)(4-ABA)] 3. The structures have an increasing degree of complexity in the overall hydrogen-bonding network. The structure of 1 comprises polymeric hydrogen-bonded chains of associated 4,4-DP and ICA molecules which propagate through a complementary site on the ICA molecules. The structure of 2 consists of two parallel polymeric hydrogen-bonded chains, each involving associated 4,4-DP and 3-ABA molecules, which cross-link through complementary 3-ABA sites. The structure of 3 is an extensive 3-dimensional hydrogen-bonded network involving all hydrogen-bond donor and acceptor sites on the constituent molecules. In each case the positions and directions of the N–H groups are important in determining the eventual lattice network.     

Increasing the sensitivity of liquid introduction mass spectrometry by combining electrospray ionization and solvent assisted inlet ionization

Combining electrospray ionization (ESI) and solvent assisted inlet ionization (SAII) provides higher ion abundances over a wide range of concentrations for peptides and proteins than either ESI or SAII. In this method, a voltage is applied to a union connector linking tubing from a solvent delivery device and the fused silica capillary, used with SAII, inserted into a heated inlet tube of an Orbitrap Exactive mass spectrometer (MS). The union can be metal or polymeric and the voltage can be applied directly or contactless. Solution flow rates from less than a 1 μL min(-1) to over 100 μL min(-1) can be accommodated. It appears that the voltage is only necessary to provide charge separation in solution, and the hot MS inlet tube and the high velocity of gas through the tube linking atmospheric pressure and vacuum provides droplet formation. As little as 100 V produces an increase in ion abundance for certain compounds using this method relative to no voltage. Interestingly, the total ion current observed with SAII and this electrosprayed inlet ionization (ESII) method are very similar for weak acid solutions, but with voltage on, the ion abundance for peptides and proteins increase as much as 100-fold relative to other compounds in the solution being analyzed. Thus, switching between SAII (voltage off) and ESII (voltage on) provides a more complete picture of the solution contents than either method alone.     

High-sensitivity ion mobility spectrometry/mass spectrometry using electrodynamic ion funnel interfaces

The utility of ion mobility spectrometry (IMS) for separation of mixtures and structural characterization of ions has been demonstrated extensively, including in biological and nanoscience contexts. A major attraction of IMS is its speed, several orders of magnitude greater than that of condensed-phase separations. Nonetheless, IMS combined with mass spectrometry (MS) has remained a niche technique, substantially because of limited sensitivity resulting from ion losses at the IMS-MS junction. We have developed a new electrospray ionization (ESI)-IMS-QTOF MS instrument that incorporates electrodynamic ion funnels at both front ESI-IMS and rear IMS-QTOF interfaces. The front funnel is of the novel "hourglass" design that efficiently accumulates ions and pulses them into the IMS drift tube. Even for drift tubes of 2-m length, ion transmission through IMS and on to QTOF is essentially lossless across the range of ion masses relevant to most applications. The rf ion focusing at the IMS terminus does not degrade IMS resolving power, which exceeds 100 (for singly charged ions) and is close to the theoretical limit. The overall sensitivity of the present ESI-IMS-MS system is comparable to that of commercial ESI-MS, which should make IMS-MS suitable for analyses of complex mixtures with ultrahigh sensitivity and exceptional throughput.     

Effect of experimental parameters on the ESI FT-ICR mass spectrum of fulvic acid

Fulvic acid (FA) is a heterogeneous mixture of organic macromolecules found in the waters, soils, and sediments of the earth's surface. The ability of electrospray ionization (ESI) to effectively transfer large ions from the solution phase to the gas phase and the coupling of ESI to the high-mass-resolution capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provide a potential method for the mass spectrometric analysis of FA. Positive- and negative-ion ESI FT-ICR MS analyses of four reference International Humic Substances Society FAs were performed. The spray solution composition was found to have a dramatic effect on the ion distributions, with high-mass aggregates (m/z approximately 2000-4000) being formed in less polar spray solutions. Positive-ion spectra for each FA obtained under optimum conditions resulted in number-average molecular weights ranging from 1700 to 1900. The mass spectra were extremely complex, with ion distributions on the order of m/z approximately 500-3000. The presence of more than one ion at each nominal mass was routinely observed. Negative-ion ESI analysis of the FA samples resulted in the observation of multiply charged ions whose distributions could be affected by the acidification of the spray solution. Solution parameters which have been reported to affect molecular weight distributions of FA such as pH, ionic strength, and concentration of multivalent cations were found to have little or no effect on the observed m/z distributions.     

Comparison of electrospray,atmospheric pressure chemical ionization,and atmospheric pressure photoionization in the identification of apomorphine,dobutamine, and entacapone phase II metabolites in biological samples

The applicability of different ionization techniques, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and a novel atmospheric pressure photoionization (APPI), were tested for the identification of the phase II metabolites of apomorphine, dobutamine, and entacapone in rat urine and in vitro incubation mixtures (rat hepatocytes and human liver microsomes). ESI proved to be the most suitable ionization method; it enabled detection of 22 conjugates, whereas APCI and APPI showed only 12 and 14 conjugates, respectively. Methyl conjugates were detected with all ionization methods. Glucuronide conjugates were ionized most efficiently with ESI. Only some of the glucuronides detected with ESI were detected with APCI and APPI. Sulfate conjugates were detected only with ESI. MS/MS experiments showed that the site of glucuronidation or sulfation could not be determined, since the primary cleavage was a loss of the conjugate group (glucuronic acid or SO3), and no site-characteristic product ions were formed. However, it may be possible to determine the site of methylation, since methylated products are more stable than glucuronides or sulfates. Furthermore, the loss of CH3 is not necessarily the primary cleavage, and site characteristic products may be formed. Identification and comparison of conjugates formed from the current model drugs were successfully analyzed in different biological specimens of common interest to biomedical research. A fairly good relation was obtained between the data from in vivo and in vitro models of drug metabolism.     

Analysis of endocrine disruptors, pharmaceuticals, and personal care products in water using liquid chromatography/tandem mass spectrometry

A method has been developed for the trace analysis of 27 compounds from a diverse group of pharmaceuticals, steroids, pesticides, and personal care products. The method employs solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), using electrospray ionization (ESI) in both positive and negative modes and atmospheric pressure chemical ionization in positive mode. Unlike many previous methods, a single SPE procedure using 1 L of water coupled to a simple LC method is used for all ionization modes. Instrument detection limits for most compounds were below 1.0 pg on column with reporting limits of 1.0 ng/L in water. Recoveries for most compounds in deionized water were greater than 80%. Sulfuric acid was found to be the preferred sample preservative, and structures of all MS/MS product ions are proposed. Matrix effects from waters with a high content of treated municipal effluent were observed in both ESI modes and are discussed in the paper.