Impact of LC-MS/MS quantification methods on analytical results by example of nationwide U.S. environmental samples.

Although different quantification methods are extensively used in environmental chemistry, the impact of the choice of method on the quality and range of analytical results is understudied. This two-part study consists of (a) in-lab evaluation and (b) a traditional meta-analysis (n = 66) of commonly used quantification methods): (i) external calibration; (ii) isotope dilution method with authentic target analogs; (iii) isotope dilution with non-target standards; and (iv) standard addition prior to LC-MS/MS in liquid chromatography tandem mass spectrometry (LC-MS/MS) by example of antibiotics in sewage sludge from across the U.S. Using method (i) as the benchmark quantification method for the antibiotic erythromycin in biosolids, other quantification methods resulted in an overestimation (110-450 %) or an underestimation (10-60 %). Using the method (iv) as the benchmark for other compounds resulted in an overestimation (101-14,700 %) or an underestimation (6-98 %). Matrix effects were also observed and were dependent on the matrix and analyte type. For example, in the case of erythromycin, all sample matrices showed signal suppression. This study showed that in the absence of isotopically labeled analogs, the most accurate alternate quantification method may need to be experimentally determined depending on the analyte. Analysis of published literature on pharmaceuticals in sewage sludge indicated that isotope dilution with authentic target analog is most commonly used, followed by non-target isotope standards, standard addition, and finally external calibration.

Volatile profiling of cacti: a preliminary assessment of the taxonomic and evolutionary significance of volatile compounds in Cylindropuntia, Grusonia, Consolea, Opuntia, Quiabentia, and Tacinga.

Volatile analyses of cacti have previously been performed on the flowers, fruits, and consumed stems. During our own investigations, we and others have observed that the cut stems of certain species of the Graveolens clade of Cylindropuntia emitted odors similar to those of rancid butter or cyanoacrylate. Some species of Consolea, Opuntia, Quiabentia, and Tacinga were found to produce similar odors. Fresh samples of Cylindropuntia and these other genera were collected, and the volatile compound profiles were analyzed by solid phase micro-extraction gas chromatography mass spectrometry. Linear discriminate analysis found the compounds to be characteristic of the odiferous cacti as the aldehydes hexanal, 2-hexenal, and nonanal; the alcohol phenethyl alcohol; the terpene ß-phellandrene; the ketone ß-ionone; and the diol 5-pentyl-1,3-benzenediol. Compounds characteristic of the non-odiferous cacti are the ketones 6-methyl-2-heptanone, 2-octanone, and 1,3-dihydro-5-methyl-2H-benzimidazol-2-one; the alkanes undecane, tridecane, pentadecane, and heptadecane; the aromatics p-cymene and 1,2,3,5-tetramethyl benzene; the esters octyl formate, methyl benzoate, and methyl salicylate; the aldehyde 2-octenal; the alcohol cyclooctyl alcohol; the imine methoxy-phenyl-oxime; the terpene 1-methyl-2-(2-propenyl)-benzene; and nine unknown compounds. Putative hybrid cacti were found to have a unique volatile profile in comparison to the parents. Additionally, differing infraspecific chromosome races, diploids (n = 11) and tetraploids (n = 22), were found to have differing volatile profiles with some compounds increasing with an increase in chromosome number while other compounds decreased with an increase in chromosome number.

Retrospective nationwide occurrence of fipronil and its degradates in U.S. wastewater and sewage sludge from 2001 - 2016.

The insecticide fipronil is under regulatory scrutiny worldwide for its toxicity to pollinators and aquatic invertebrates. We conducted the first U.S. nationwide, longitudinal study of sewage sludges for fiproles, i.e., the sum of fipronil and its major degradates (fipronil sulfone, sulfide, amide, and desulfinyl). Archived sludges (n = 109) collected in three campaigns over 15 years were analyzed by isotope dilution liquid chromatography tandem mass spectrometry, revealing ubiquitous fiprole occurrence (0.2-385.3 µg/kg) since 2001 and a significant increase (2.4 ±â€¯0.3 fold; p < 0.005) both from 2001 to 2006/7 and from 2001 to 2015/6, but not a significant increase from 2006/7 to 2015/6 (p = 0.275). A geospatial analysis showed fiprole levels in municipal sludges to be uncoupled from agricultural use of fipronil on cropland surrounding sampled municipalities, thus pointing to non-agricultural uses (i.e., spot-on treatment and urban pest control) as a major source of fiprole loading to wastewater. Whereas anaerobic digestion was correlated with increases in fipronil sulfide at the expense of parental fipronil (p < 0.001), total fiprole levels in sewage sludges were similar regardless of the solids treatment approach applied (p = 0.519). Treatment plant effluent available from 12 facilities in 2015/6 contained fiproles at 0.3-112.9 ng/L, exceeding the United States Environmental Protection Agency (USEPA) aquatic invertebrate life benchmark for chronic fipronil exposure (11 ng/L) in 67% of cases. Whereas the USEPA identified fipronil in sludge only recently (2015), retrospective analyses and modeling conducted here show contaminant ubiquity and nationwide increases of fiprole mass (compared to 2001 levels) in U.S. municipal sludge (1140 ±â€¯230 kg in 2015/6), and treated effluent nationwide (1970 ±â€¯390 kg in 2015/6) over the past 15 years.

U.S. nationwide reconnaissance of ten infrequently monitored antibiotics in municipal biosolids.

Ten infrequently monitored antibiotics in biosolids were examined in archived American sewage sludges (n = 79) collected as part of the 2006/2007 U.S. Environmental Protection Agency (EPA) Targeted National Sewage Sludge Survey. This study inspected the occurrence of amoxicillin, ampicillin, erythromycin, furazolidone [proxy metabolite: 3-(2-nitrobenzylidenamino)-2-oxazolidinone (NP-AOZ)], nalidixic acid, oxolinic acid, oxytetracycline, spiramycin, sulfadimidine, and sulfadimethoxine in sewage sludges after nearly a decade in frozen storage. Six antibiotics were detected at the following average concentrations (ng/g dry weight): amoxicillin (1.0), nalidixic acid (19.1), oxolinic acid (2.7), erythromycin (0.6), oxytetracycline (4.5), and ampicillin (14.8). The remaining four were not detected in any samples (

Ion Mobility Spectrometry-Mass Spectrometry Coupled with Gas-Phase Hydrogen/Deuterium Exchange for Metabolomics Analyses.

Ion mobility spectrometry-mass spectrometry (IMS-MS) in combination with gas-phase hydrogen/deuterium exchange (HDX) and collision-induced dissociation (CID) is evaluated as an analytical method for small-molecule standard and mixture characterization. Experiments show that compound ions exhibit unique HDX reactivities that can be used to distinguish different species. Additionally, it is shown that gas-phase HDX kinetics can be exploited to provide even further distinguishing capabilities by using different partial pressures of reagent gas. The relative HDX reactivity of a wide variety of molecules is discussed in light of the various molecular structures. Additionally, hydrogen accessibility scoring (HAS) and HDX kinetics modeling of candidate (in silico) ion structures is utilized to estimate the relative ion conformer populations giving rise to specific HDX behavior. These data interpretation methods are discussed with a focus on developing predictive tools for HDX behavior. Finally, an example is provided in which ion mobility information is supplemented with HDX reactivity data to aid identification efforts of compounds in a metabolite extract. Graphical Abstract ᅟ.

Ion Mobility, Hydrogen/Deuterium Exchange, and Isotope Scrambling: Tools to Aid Compound Identification in 'Omics Mixtures.

Liquid chromatography tandem mass spectrometry (LC-MS/MS), a widely used method for comparative 'omics analysis, experiences challenges with compound identification due to matrix effects, difficulty in separating isomer and isobaric ions, and long analysis times. Ion mobility spectrometry (IMS) has proven to be useful in separating isomer and isobar ions; however, IMS-MS suffers from decreased peak capacity due to the correlation in ion size and mass. In proof-of-principle experiments, the use of gas-phase hydrogen/deuterium exchange (HDX) combined with IMS-MS/MS techniques is demonstrated to offer advantages for compound identification. Measurements providing unique information for ions include m/z value, drift time in He buffer gas, drift time in He and D2O buffer gases, deuterium incorporation pattern (isotopic distribution), deuterium incorporation pattern after collisional activation, and fragment ion deuterium incorporation pattern upon collision-induced dissociation (CID). These techniques are here shown to be highly reproducible (drift time coefficients of variation < 1.0% and isotopic pattern root-mean-square deviations of 0.5-1.5%) while demonstrating an increased ability to distinguish individual molecules from diverse classes of compounds (peptides, catecholamines, nucleosides, amino acids, etc.). The concept of using such (and similar) information for rapid, high-throughput molecular identification via database searching of standard libraries is briefly discussed, and an example of such usage is presented for a bonafide metabolite extract sample.

Comparative plasma proteomic studies of pulmonary TiO2 nanoparticle exposure in rats using liquid chromatography tandem mass spectrometry.

Mounting evidence suggests that pulmonary exposure to nanoparticles (NPs) has a toxic effect on biological systems. A number of studies have shown that exposure to NPs result in systemic inflammatory response, oxidative stress, and leukocyte adhesion. However, significant knowledge gaps exist for understanding the key molecular mechanisms responsible for altered microvasculature function. Utilizing comprehensive LC-MS/MS and comparative proteomic analysis strategies, important proteins related to TiO2 NP exposure in rat plasma have been identified. Molecular pathway analysis of these proteins revealed 13 canonical pathways as being significant (p ≤ 0.05), but none were found to be significantly up or down-regulated (z>|2|). This work lays the foundation for future research that will monitor relative changes in protein abundance in plasma and tissue as a function of post-exposure time and TiO2 NP dosage to further elucidate mechanisms of pathway activation as well as to decipher other affected pathways.

Advances in ion mobility-mass spectrometry instrumentation and techniques for characterizing structural heterogeneity.

Over the last decade, the field of ion mobility-mass spectrometry (IM-MS) has experienced dramatic growth in its application toward ion structure characterization. Enabling advances in instrumentation during this time period include improved conformation resolution and ion sensitivity. Such advances have rendered IM-MS a powerful approach for characterizing samples presenting a diverse array of ion structures. The structural heterogeneity that can be interrogated by IM-MS techniques now ranges from samples containing mixtures of small molecules exhibiting a variety of structural types to those containing very large protein complexes and subcomplexes. In addition to this diversity, IM-MS techniques have been used to probe spontaneous and induced structural transformations occurring in solution or the gas phase. To support these measurement efforts, significant advances have been made in theoretical methods aimed at translating IM-MS data into structural information. These efforts have ranged from providing more reliable trial structures for comparison to the experimental measurements to dramatically reducing the time required to calculate collision cross sections for such structures. In this short review, recent advances in developments in IM-MS instrumentation, techniques, and theory are discussed with regard to their implications for characterization of gas- and solution-phase structural heterogeneity.

Huntingtin N-Terminal Monomeric and Multimeric Structures Destabilized by Covalent Modification of Heteroatomic Residues.

Early stage oligomer formation of the huntingtin protein may be driven by self-association of the 17-residue amphipathic α-helix at the protein's N-terminus (Nt17). Oligomeric structures have been implicated in neuronal toxicity and may represent important neurotoxic species in Huntington's disease. Therefore, a residue-specific structural characterization of Nt17 is crucial to understanding and potentially inhibiting oligomer formation. Native electrospray ion mobility spectrometry-mass spectrometry (IMS-MS) techniques and molecular dynamics simulations (MDS) have been applied to study coexisting monomer and multimer conformations of Nt17, independent of the remainder of huntingtin exon 1. MDS suggests gas-phase monomer ion structures comprise a helix-turn-coil configuration and a helix-extended-coil region. Elongated dimer species comprise partially helical monomers arranged in an antiparallel geometry. This stacked helical bundle may represent the earliest stages of Nt17-driven oligomer formation. Nt17 monomers and multimers have been further probed using diethylpyrocarbonate (DEPC). An N-terminal site (N-terminus of Threonine-3) and Lysine-6 are modified at higher DEPC concentrations, which led to the formation of an intermediate monomer structure. These modifications resulted in decreased extended monomer ion conformers, as well as a reduction in multimer formation. From the MDS experiments for the dimer ions, Lys6 residues in both monomer constituents interact with Ser16 and Glu12 residues on adjacent peptides; therefore, the decrease in multimer formation could result from disruption of these or similar interactions. This work provides a structurally selective model from which to study Nt17 self-association and provides critical insight toward Nt17 multimerization and, possibly, the early stages of huntingtin exon 1 aggregation.

An improved UHPLC-UV method for separation and quantification of carotenoids in vegetable crops.

Carotenoid identification and quantitation is critical for the development of improved nutrition plant varieties. Industrial analysis of carotenoids is typically carried out on multiple crops with potentially thousands of samples per crop, placing critical needs on speed and broad utility of the analytical methods. Current chromatographic methods for carotenoid analysis have had limited industrial application due to their low throughput, requiring up to 60 min for complete separation of all compounds. We have developed an improved UHPLC-UV method that resolves all major carotenoids found in broccoli (Brassica oleracea L. var. italica), carrot (Daucus carota), corn (Zea mays), and tomato (Solanum lycopersicum). The chromatographic method is completed in 13.5 min allowing for the resolution of the 11 carotenoids of interest, including the structural isomers lutein/zeaxanthin and α-/ß-carotene. Additional minor carotenoids have also been separated and identified with this method, demonstrating the utility of this method across major commercial food crops.