Quantitative analysis of methyl and propyl parabens in neonatal DBS using LC–MS/MS

Aim: Excipients are used to overcome the chemical, physical and microbiological challenges posed by developing formulated medicines. Both methyl and propyl paraben are commonly used in pediatric liquid formulations. There is no data on systemic exposure to parabens in neonates. The European Study of Neonatal Exposure to Excipients project has investigated this. Results & methodology: DBS sampling was used to collect opportunistic blood samples. Parabens were extracted from the DBS and analyzed using a validated LC-MS/MS assay.

Discussion & Conclusion: The above assay was applied to analyze neonatal DBS samples. The blood concentrations of parabens in neonates confirm systemic exposure to parabens following administration of routine medicines.

Desenvolvimento e validação de metodologia analítica para a determinação de fármacos em amostyras de água, superficial e tratada, utilizando a cromatografia líquida de ultra performance acoplada a espectrometria de massas tandem (UPLC-MS/MS)

Dissertação (Mestrado)

Seasonal and Species Variation of the Hepatotoxin Indospicine in Australian Indigofera Legumes as Measured by UPLC−MS/MS

Livestock industries have maintained a keen interest in pasture legumes because of the high protein content and nutritive value. Leguminous Indigofera plant species have been considered as having high feeding values to be utilized as pasture, but the occurrence of the toxic constituent indospicine in some species has restricted this utility. Indospicine has caused both primary and secondary hepatotoxicosis and also reproductive losses, but has only previously been determined in a small number of Indigofera species. This paper validates a high throughput ultra-performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS) method to determine indospicine content of various Indigofera species found in Australian pasture. Twelve species of Indigofera together with Indigastrum parviflorum plants were collected and analysed. Out of the 84 samples analyzed, *I. spicata contained the highest indospicine level (1003 ± 328 mg/kg DM, n = 4) followed by I. linnaei (755 ± 490 mg/kg DM, n = 51). Indospicine was not detected in 9 of the remaining 11 species, and at only low levels (<10 mg/kg DM) in 2 out of 8 I. colutea specimens and in 1 out of 5 I. linifolia specimens. Indospicine concentrations were below quantitation levels for other Indigofera spp. (I. adesmiifolia, I. georgei, I. hirsuta, I. leucotricha,* I. oblongifolia, I. australis and I. trita) and Indigastrum parviflorum. One of the more significant findings to emerge from this study is that the indospicine content of I. linnaei is highly variable (159 to 2128 mg/kg DM, n = 51), and differs across both regions and seasons. Its first re-growth after spring rain has a higher (p < 0.01) indospicine content than growth following more substantial summer rain. The species collected include the predominant Indigofera in Australia pasture, and of these, only *I. spicata and I. linnaei contain high enough levels of indospicine to pose a potential toxic threat to grazing herbivores.

CHARACTERIZATION OF POLYUBIQUITIN CHAINS BY MASS SPECTROMETRY

The work outlined in this dissertation will allow biochemists and cellular biologists to characterize polyubiquitin chains involved in their cellular environment by following a facile mass spectrometric based workflow. The characterization of polyubiquitin chains has been of interest since their discovery in 1984. The profound effects of ubiquitination on the movement and processing of cellular proteins depend exclusively on the structures of mono and polyubiquitin modifications anchored or unanchored on the protein within the cellular environment. However, structure-function studies have been hindered by the difficulty in identifying complex chain structures due to limited instrument capabilities of the past. Genetic mutations or reiterative immunoprecipitations have been used previously to characterize the polyubiquitin chains, but their tedium makes it difficult to study a broad ubiquitinome. Top-down and middle-out mass spectral based proteomic studies have been reported for polyubiquitin and have had success in characterizing parts of the chain, but no method to date has been successful at differentiating all theoretical ubiquitin chain isomers (ubiquitin chain lengths from dimer to tetramer alone have 1340 possible isomers). The workflow presented here can identify chain length, topology and linkages present using a chromatographic-time-scale compatible, LC-MS/MS based workflow. To accomplish this feat, the strategy had to exploit the most recent advances in top-down mass spectrometry. This included the most advanced electron transfer dissociation (ETD) activation and sensitivity for large masses from the orbitrap Fusion Lumos. The spectral interpretation had to be done manually with the aid of a graphical interface to assign mass shifts because of a lack of software capable to interpret fragmentation across isopeptide linkages. However, the method outlined can be applied to any mass spectral based system granted it results in extensive fragmentation across the polyubiquitin chain; making this method adaptable to future advances in the field.

INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS

Exosomes released by myeloid-derived suppressor cells (MDSC) are 30 nm in diameter extracellular vesicles that have been shown to carry biologically active proteins as well as ubiquitin molecules. Ubiquitin is known to have many functions, including involvement in the formation of exosomes, although the exact role is highly contested. In the study reported here, the proteome and ubiquitome of MDSC exosomes has been investigated by bottom-up proteomics techniques. This report identifies more than 1000 proteins contained in the MDSC exosome cargo and 489 sites of ubiquitination in more than 300 ubiquitinated proteins based on recognition of glycinylglycine tagged peptides without antibody enrichment. This has allowed extensive chemical and biological characterization of the ubiquitinated cohort compared to that of the entire protein cargo to support hypotheses on the role of ubiquitin in exosomes.

Desenvolvimento e validação de métodos para a determinação de agrotóxicos em água e solo das áreas de recarga de Aquífero Guarani, na região das nascentes do Rio Araguaia, MT/GO.

Abstract: The area near the Araguaia River, between Goiás and Mato Grosso States, is the location of a portion of the recharging of the Guarani Aquifer, which is one of the world¿s largest aquifer systems and an important source of drinking water. This reservoir could be threatened by the widespread use of pesticides in maize and soybean cultivation in this area. Thus, this work developed analytical methods for the determination of imazethapyr, nicosulfuron, imazaquin, carbofuran, atrazine, linuron, clorimuronethyl and diflubenzuron, pesticides used in maize and soybean cultivation. Pesticide separation, identification and quantification were performed using High-Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD) and Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). Solid Phase Extraction (SPE) with C18 sorbents was optimized for sample extraction from water. Soil samples were extracted by mechanical shaking, sonication or microwave-assisted extraction with industrial and home microwave ovens. Methods were validated resulting in limits of quantification (LOQ) for the pesticides in water in the range of 0.015-0.1 ng mL, using SPE-HPLC-DAD, and 0.01 ng mL using LC-ESI-MS/MS. LOQ of 1 ng mL for all pesticides in soil were achieved using the home microwave oven and LC-ESI-MS/MS. Recoveries for pesticides with all methods were in the range 70-120 %. Relative standard deviations for repeatability and intermediate precision were less than 15 %. SPEHPLC- DAD and LC-ESI-MS/MS were employed for the analysis of samples of water from the recharge area and most of the pesticides were detected at concentrations below the minimum residue limit (MRL) of 0.1 ng mL established by the European Community. The home microwave oven and LC-ESI-MS/MS were used for the analysis of soil samples from two other regions of Brazil and the pesticides were not detected in these samples. Adsorption and desorption parameters were determined for imazethapyr, imazaquin, nicosulfuron and chlorimuron-ethyl, indicating that these pesticides have little affinity for the soil of the region of the Guarani Aquifer recharge, and show significant leaching potential, according to the ground water ubiquity score (GUS index) for these pesticides.

Liquid chromatographic-mass spectrometric method for determination of drug content uniformity of two commonly used dermatology medications in a split-tablet dosage form

Purpose: To develop and validate a simple, efficient and reliable Liquid chromatographic-mass spectrometric (LC-MS/MS) method for the quantitative determination of two dermatological drugs, Lamisil® (terbinafine) and Proscar® (finasteride), in split tablet dosage form. Methods: Thirty tablets each of the 2 studied medications were randomly selected. Tablets were weighed and divided into 3 groups. Ten tablets of each drug were kept intact, another group of 10 tablets were manually split into halves using a tablet cutter and weighed with an analytical balance; a third group were split into quarters and weighed. All intact and split tablets were individually dissolved in a water: methanol mixture (4:1), sonicated, filtered and further diluted with mobile phase. Optimal chromatographic separation and mass spectrometric detection were achieved using an Agilent 1200 HPLC system coupled with an Agilent 6410 triple quadrupole mass spectrometer. Analytes were eluted through an Agilent eclipse plus C8 analytical column (150 mm × 4.6 mm, 5 μm) with a mobile phase composed of solvent A (water) containing 0.1% formic acid and 5mM ammonium formate pH 7.5, and solvent B (acetonitrile mixed with water in a ratio A:B 55:45) at a flow rate of 0.8 mL min-1 with a total run time of 12 min. Mass spectrometric detection was carried out using positive ionization mode with analyte quantitation monitored by multiple reaction monitoring (MRM) mode. Results: The proposed analytical method proved to be specific, robust and adequately sensitive. The results showed a good linear fit over the concentration range of 20 - 100 ng mL-1 for both analytes, with a correlation coefficient (r2) ≥ 0.999 and 0.998 for finasteride and terbinafine, respectively. Following tablet splitting, the drug content of the split tablets fell outside of the proxy USP specification for at least 14 halves (70 %) and 34 quarters (85 %) of FIN, as well as 16 halves (80 %) and 37 quarters (92.5 %) of TBN. Mean weight loss, after splitting, was 0.58 and 2.22 % for FIN half- and quarter tablets, respectively, and 3.96 and 4.09 % for TBN half- and quarter tablets,respectively. Conclusion: The proposed LC-MS/MS method has successfully been used to provide precise drug content uniformity of split tablets of FIN and TBN. Unequal distribution of the drug on the split tablets is indicated by the high standard deviation beyond the accepted value. Hence, it is recommended not to split non-scored tablets especially, for those medications with significant toxicity

Comparison of the binding stoichiometries of positively charged DNA-binding drugs using positive and negative ion electrospray ionization mass spectrometry

Positive and negative ion electrospray ionization (ESI) mass spectra of complexes of positively charged small molecules (distamycin, Hoechst 33258, [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2) have been compared. [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2 bind to DNA by intercalation. Negative ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA showed ions from DNA-ligand complexes consistent with solution studies. In contrast, only ions from freeDNAwere present in positive ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA, highlighting the need for obtaining ESI mass spectra of non-covalent complexes under a range of experimental conditions. Negative ion spectra of mixtures of the minor groove binder Hoechst 33258 with DNA containing a known minor groove binding sequence were dominated by ions from a 1:1 complex. In contrast, in positive ion spectra there were also ions present from a 2:1 (Hoechst 33258: DNA) complex, suggesting an alternative binding mode was possible either in solution or in the gas phase. When Hoechst 33258 was mixed with a DNA sequence lacking a high affinity minor groove binding site, the negative ion ESI mass spectra showed that 1:1 and 2:1 complexes were formed, consistent with existence of binding modes other than minor groove binding. The data presented suggest that comparison of positive and negative ion ESI-MS spectra might provide an insight into various binding modes in both solution and the gas phase.

Addressing accuracy and precision issues in iTRAQ quantitation

iTRAQ (isobaric tags for relative or absolute quantitation) is a mass spectrometry technology that allows quantitative comparison of protein abundance by measuring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation during MS/MS. However, current data analysis techniques for iTRAQ struggle to report reliable relative protein abundance estimates and suffer with problems of precision and accuracy. The precision of the data is affected by variance heterogeneity: low signal data have higher relative variability; however, low abundance peptides dominate data sets. Accuracy is compromised as ratios are compressed toward 1, leading to underestimation of the ratio. This study investigated both issues and proposed a methodology that combines the peptide measurements to give a robust protein estimate even when the data for the protein are sparse or at low intensity. Our data indicated that ratio compression arises from contamination during precursor ion selection, which occurs at a consistent proportion within an experiment and thus results in a linear relationship between expected and observed ratios. We proposed that a correction factor can be calculated from spiked proteins at known ratios. Then we demonstrated that variance heterogeneity is present in iTRAQ data sets irrespective of the analytical packages, LC-MS/MS instrumentation, and iTRAQ labeling kit (4-plex or 8-plex) used. We proposed using an additive-multiplicative error model for peak intensities in MS/MS quantitation and demonstrated that a variance-stabilizing normalization is able to address the error structure and stabilize the variance across the entire intensity range. The resulting uniform variance structure simplifies the downstream analysis. Heterogeneity of variance consistent with an additive-multiplicative model has been reported in other MS-based quantitation including fields outside of proteomics; consequently the variance-stabilizing normalization methodology has the potential to increase the capabilities of MS in quantitation across diverse areas of biology and chemistry.

Desorption electrospray ionisation mass spectrometry of stabilised polyesters reveals activation of hindered amine light stabilisers

The use of hindered amine light stabilizers (HALS) to retard thermo- and photo-degradation of polymers has become increasingly common. Proposed mechanisms of polymer stabilisation involve significant changes to the HALS chemical structure; however, reports of the characterisation of these modified chemical species are limited. To better understand the fate of HALS and determine their in situ modifications, desorption electrospray ionisation mass spectrometry (DESI-MS) was employed to characterise ten commercially available HALS present in polyester-based coil coatings. TINUVIN® 770, 292, 144, 123, 152, and NOR371; HOSTAVIN® 3052, 3055, 3050, and 3058 were separately formulated with a pigmented, thermosetting polyester resin, cured on metal at 262 C and analysed directly by DESI-MS. High-level ab initio molecular orbital theory calculations were also undertaken to aid the mechanistic interpretation of the results. For HALS containing N-substituted piperidines (i.e., N-CH3, N-C(O)CH3, and N-OR) a secondary piperidine (N-H) analogue was detected in all cases. The formation of these intermediates can be explained either through hydrogen abstraction based mechanisms or direct N-OR homolysis with the former dominant under normal service temperatures (ca. 25-80 C), and the latter potentially becoming competitive under the high temperatures associated with curing (ca. 230-260 C). © 2013 Elsevier Ltd. All rights reserved.

Elucidation of double bond position in unsaturated lipids by ozone electrospray ionization mass spectrometry

The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids, however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication \[Thomas, M. C.; Mitchell, T. W.; Blanksby, S. J. J. Am. Chem. Soc. 2006, 128, 58-59], we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including acidic and neutral glycerophospholipids, sphingomyelins, and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges, or the adduction (e.g., \[M - H](-), \[M - 2H](2-), \[M + H](+), \[M + Na](+), \[M + NH4](+)). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio (m/z). The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used, in conjunction with conventional tandem mass spectral analysis, for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely, SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1), and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and, when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.

Desorption electrospray ionisation mass spectrometry reveals in situ modification of a hindered amine light stabiliser resulting from direct N–OR bond cleavage

Detection and characterisation of structural modifications of a hindered amine light stabiliser (HALS) directly from a polyester-based coil coating have been achieved by desorption electrospray ionisation mass spectrometry (DESI-MS) for the first time. In situ detection is made possible by exposing the coating to an acetone vapour atmosphere prior to analysis. This is a gentle and non-destructive treatment that allows diffusion of analyte to the surface without promoting lateral migration. Using this approach a major structural modification of the HALS TINUVIN®123 (bis(1-octyloxy-2,2,6,6-tetramethyl-4-piperidyl) sebacate) was discovered where one N-ether piperidine moiety (N-OC8H17) is converted to a secondary piperidine (N–H). With the use of 2-dimensional DESI-MS imaging the modification was observed to arise during high curing temperatures (ca. 260 °C) and under simulated physiological conditions (80 °C, full solar spectrum). It is proposed that the secondary piperidine derivative is a result of a highly reactive aminyl radical intermediate produced by N–O homolytic bond cleavage. The nature of the bond cleavage is also suggested by ESR spin-trapping experiments employing α-phenyl-N-tert-butyl nitrone (PBN) in toluene at 80 °C. The presence of a secondary piperidine derivative in situ and the implication of N–OR competing with NO–R bond cleavage suggest an alternative pathway for generation of the nitroxyl radical—an essential requirement in anti-oxidant activity that has not previously been described for the N-ether sub-class of HALS.

Structural identification of hindered amine light stabilisers in coil coatings using electrospray ionisation tandem mass spectrometry

Hindered amine light stabilisers (HALS) are the most effective antioxidants currently available for polymer systems in post-production, in-service applications, yet the mechanism of their action is still not fully understood. Structural characterisation of HALS in polymer matrices, particularly the identification of structural modifications brought about by oxidative conditions, is critical to aid mechanistic understanding of the prophylactic effects of these molecules. In this work, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) was applied to the analysis of a suite of commercially available 2,2,6,6-tetramethylpiperidine-based HALS. Fragmentation mechanisms for the \[M + H](+) ions are proposed, which provide a rationale for the product ions observed in the MS/MS and MS(3) mass spectra of N-H, N-CH(3), N-C(O)CH(3) and N-OR containing HALS (where R is an alkyl substituent). A common product ion at m/z 123 was identified for the group of antioxidants containing N-H, N-CH3 or N-C(0)CH3 functionality, and this product ion was employed in precursor ion scans on a triple quadrupole mass spectrometer to identify the HALS species present in a crude extract from of a polyester-based coil coating. Using MS/MS, two degradation products were unambiguously identified. This technique provides a simple and selective approach to monitoring HALS structures within complex matrices. Copyright (C) 2010 John Wiley & Sons, Ltd.

Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry

Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag]+ and [PC (18:1/16:0) + Ag]+} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation.

Effects of mesostructured silica catalysts on the depolymerization of organosolv lignin fractionated from woody eucalyptus

Isolated and purified organosolv eucalyptus wood lignin was depolymerized at different temperatures with and without mesostructured silica catalysts (i.e., SBA-15, MCM-41, ZrO2-SBA-15 and ZrO2-MCM-41). It was found that at 300 oC for 1 h with a solid/liquid ratio of 0.0175/1 (w/v), the SBA-15 catalyst with high acidity gave the highest syringol yield of 23.0% in a methanol/water mixture (50/50, wt/wt). Doping with ZrO2 over these catalysts did not increase syringol yield, but increased the total amount of solid residue. Gas chromatography-mass spectrometry (GC-MS) also identified other main phenolic compounds such as 1-(4-hydroxy-3,5-dimethoxyphenyl)-ethanone, 1,2-benzenediol, and 4-hydroxy-3,5-dimethoxy-benzaldehyde. Analysis of the lignin residues with Fourier transform-Infrared spectroscopy (FT-IR) indicated decreases in the absorption bands intensities of OH group, C-O stretching of syringyl ring and aromatic C-H deformation of syringol unit, and an increase in band intensities associated with the guaiacyl ring, confirming the type of products formed.