Evaluation of mycotoxins and their metabolites in human breast milk using liquid chromatography coupled to high resolution mass spectrometry

Humans can be exposed to mycotoxins through the food chain. Mycotoxins are mainly found as contaminants in food and could be subsequently excreted via biological fluids such as urine or human breast milk in native or metabolised form. Since breast milk is usually supposed as the only food for new-borns, the occurrence of mycotoxins in thirty-five human milk samples was evaluated by a newly developed method based on QuEChERS extraction and UHPLC–HRMS detection. The method described here allows the detection of target mycotoxins in order to determine the quality of this initial feeding. The method has been fully validated, with recoveries ranging from 64% to 93% and relative standard deviations (RSD, %) being lower than 20%. Using the method described, non-metabolised mycotoxins such as ZEA, NEO, NIV, ENA, ENA₁, ENB, ENB₁ and metabolites, such as ZEA metabolites, HT-2, DOM and T-2 triol were detected in human milk samples. Results obtained help to estimate the exposure of mothers and infants to mycotoxins. Moreover, to the best of our knowledge, this is the first work describing the simultaneous detection, quantification and screening of mycotoxins and their metabolites in human mature milk.     

Mass spectrometry and planetary exploration: A brief review and future projection

Since the inception of mass spectrometry more than a century ago, the field has matured as analytical capabilities have progressed, instrument configurations multiplied, and applications proliferated. Modern systems are able to characterize volatile and nonvolatile sample materials, quantitatively measure abundances of molecular and elemental species with low limits of detection, and determine isotopic compositions with high degrees of precision and accuracy. Consequently, mass spectrometers have a rich history and promising future in planetary exploration. Here, we provide a short review on the development of mass analyzers and supporting subsystems (eg, ionization sources and detector assemblies) that have significant heritage in spaceflight applications, and we introduce a selection of emerging technologies that may enable new and/or augmented mission concepts in the coming decades.     

Normalization techniques for high-throughput screening by infrared matrix-assisted laser desorption electrospray ionization mass spectrometry

Mass spectrometry (MS) is an effective analytical tool for high-throughput screening (HTS) in the drug discovery field. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MS is a high-throughput platform that has achieved analysis times of sub-seconds-per-sample. Due to the high-throughput analysis speed, methods are needed to increase the analyte signal while decreasing the variability in IR-MALDESI-MS analyses to improve data quality and reduce false-positive hits. The Z-factor is used as a statistic of assay quality that can be improved by reducing the variation of target ion abundances or increasing signal. Herein we report optimal solvent compositions for increasing measured analyte abundances with direct analysis by IR-MALDESI-MS. We also evaluate normalization strategies, such as adding a normalization standard that is similar or dissimilar in structure to the model target drug, to reduce the variability of measured analyte abundances with direct analyses by IR-MALDESI-MS in both positive and negative ionization modes.     

新型亲和去垢小柱净化-液相色谱-串联质谱法分析水稻叶片蛋白质组

采用亲和去垢小柱净化,建立了水稻叶片蛋白质组的纳升液相色谱-串联质谱分析方法。水稻叶片蛋白质分别采用酚提取法结合十二烷基硫酸钠(sodium dodecyl sulfate,SDS)裂解,裂解液经亲和去垢小柱净化,酶解肽段用纳升液相色谱-线性离子阱/静电场轨道阱组合式高分辨质谱(nanoLC-LTQ/Orbitrap MS)分析,相关数据库检索鉴定。比较了超滤辅助样品制备法(FASP法)、亲和去垢小柱法和丙酮沉淀法对SDS去除效率及对蛋白质鉴定结果的影响。结果表明:3种方法均有较好的SDS去除效果(去除效率均大于95%);尽管3种方法鉴定的蛋白质种类具有一定的互补性,但以亲和去垢小柱法鉴定的蛋白质数目最多,为563种,远多于FASP法和丙酮沉淀法的196和306种;此外,亲和去垢小柱法适合于各种相对分子质量和不同pI值蛋白质的净化,而FASP法和丙酮沉淀法中不同相对分子质量和pI值蛋白质均有类似程度的损失。采用本文建立的方法,一次进样分析可鉴定出水稻叶片蛋白质多达588种;肽段匹配数≥2的296个蛋白质的生物学功能主要分为结合活性、酶活性、转移运输活性和结构组成等。该蛋白质分析方法可为开展水稻蛋白质组学研究提供技术参考。     

Structural characterization of in vitro rat liver microsomal metabolites of antihistamine desloratadine using LTQ-Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange HR-LC/MS

In vitro drug metabolism study is an integral part of drug discovery process. In this report, we have described the application of LTQ-Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange high resolution (HR)-LC/MS for structural characterization of in vitro rat liver microsomal metabolites of antihistamine desloratadine. Five metabolites M1--M5 have been identified, including three hydroxylated metabolites M1--M3, one N-oxide M4 and one uncommon aromatized N-oxide M5. Accurate mass data have been obtained in both full scan and MSn mode support assignments of metabolite structures with reported mass errors less than 3 ppm. Online H/D exchange HR-LC/MS experiments provide additional evidence in differentiating hydroxylated metabolites from N-oxides. This study demonstrates the effectiveness of this approach in structural characterization of drug metabolites.     

Metabolism of TM‐2, a potential antitumor drug,in rats by using LC–MS

TM‐2 (13‐(N‐Boc‐3‐i‐butylisoserinoyl‐4,10‐β‐diacetoxy‐2‐α‐benzoyloxy‐5‐β‐20‐epoxy‐1,13‐α‐dihydroxy‐9‐oxo‐19‐norcyclopropa[g]tax‐11‐ene) is a novel semisynthetic taxane derivative. Our previous study suggested that TM‐2 is a promising antitumor analogue. In this paper, the metabolism of TM‐2 was investigated in rats following intravenous administration. Two different types of mass spectrometry—hybrid linear trap quadrupole orbitrap (LTQ‐Orbitrap) mass spectrometry and triple‐quadrupole tandem (QQQ) mass spectrometry—were employed to acquire structural information of TM‐2 metabolites. A total of 17 components were identified as the metabolites of TM‐2 in bile, feces, and urine samples. Accurate mass measurement using LC–LTQ‐Orbitrap‐MS was used to determine the accurate mass data and elemental composition of metabolites thereby confirming the proposed structures of the metabolites. The metabolites proposed were mainly oxidates of TM‐2, including methoxy, hydroxyl, dihydroxy, and trihydroxyl analogues. The major metabolic pathway of TM‐2 was the hydroxylation of the taxane ring or the lateral chain. These important metabolic data serve as a useful resource to support further research of TM‐2.     

Size and concentration determination of (functionalised) fullerenes in surface and sewage water matrices using field flow fractionation coupled to an online accurate mass spectrometer: Method development and validation

In order to assess the environmental risks of a compound it is imperative to have suitable and reliable techniques for its determination in environmental matrices. In this paper, we focused on a method development for the recently introduced online coupling of a field flow fractionation (FFF) system to an Orbitrap-HRMS, that allows the simultaneous size and concentration determination of different aqueous fullerene aggregates and their concentrations in different size fractions. A 0.05% NH₄OH solution in water was identified as the best carrier liquid for the analysis of the three different aqueous fullerene suspensions (C₆₀ [60], [6,6]-phenyl-C₆₁ butyric acid methyl ester ([60]PCBM) and [6,6]-(bis)phenyl-C₆₁ butyric acid methyl ester ([60]bisPCBM)). The multi-angle light scattering (MALS) data received after employing the ammonia solution was consistent with both the theory and calibration using well defined Au and latex particles. The LODs obtained using Orbitrap HRMS detection were 0.1 μg L⁻¹ for an injection volume of 100 μL which are significantly better than the LODs obtained by using UV (20 μg L⁻¹) and MALS detectors (5 μg L⁻¹). However, these LODs can be further improved as in theory there is no limit to the amount of sample that can be injected into the FFF. Environmental samples (river and sewage water) were spiked with fullerenes and the fractograms obtained for these samples revealed that the matrix does affect the size of fullerene aggregates. Information on the size distribution can be useful for the risk assessment of these particles.     

A high‐throughput glutathione trapping assay with combined high sensitivity and specificity in high‐resolution mass spectrometry by applying product ion extraction and data‐dependent neutral loss

Reactive metabolites are thought to play a pivotal role in the pathogenesis of some drug‐induced liver injury (DILI) and idiosyncratic adverse drug reactions (IADRs), which is of concern to patient safety and has been a cause of drugs being withdrawn from the market place. To identify drugs with a lower propensity for causing DILI and/or IADRs, high‐throughput assays to capture reactive metabolites are required in pharmaceutical industry for early drug discovery risk assessment. We describe the development of an assay to detect glutathione adducts with combined high sensitivity, enhanced specificity, and rapid data analysis. In this assay, compounds were incubated with human liver microsomes and a mixture of 1:1 of GSH (γ‐GluCysGly): GSX(γ‐GluCysGly‐¹³C₂¹⁵N) in a 96‐well plate format. UPLC‐UV and LTQ Orbitrap XL were employed to detect GSH‐adducts using the following mass spectrometry setups: (a) selected ion monitoring (SIM) at m/z of 274 ± 3 Da in negative mode with in‐source fragmentation (SCID), which enables simultaneously monitoring two characteristic product ions of m/z 272.0888 (γ‐glutamyl‐dehydroalanyl‐glycine) and 275.0926 (γ‐glutamyl‐dehydroalanyl‐glycine‐¹³C₂¹⁵N); (b) full scan mode for acquisition of exact mass of glutathione adducts; (c) data‐dependent MS² scan through isotopic matching (M:M + 3.00375 = 1:1) for monitoring neutral loss fragments (144 Da from dehydroalanyl‐glycine) and for structural information of glutathione adducts. This approach was qualified using eight compounds known to form GSH conjugates as reported in the literature. The high sensitivity and specificity were demonstrated in identifying unique CysGly adducts in the case of clozapine, diclofenac, and raloxifene and in identifying GSH‐adducts of fragmented parent molecules in the case of amodiaquine and troglitazone. In addition, LC‐UV chromatograms in the presence or absence of GSH/GSX allowed for identification of the rearranged glutathione adducts without aforementioned characteristic fragment ions. Implement of this assay in drug discovery small molecule programs has successfully guided drug design.