Shotgun metabolomic profiles in maternal urine identify potential mass spectral markers of abnormal fetal biochemistry – dihydrouracil and progesterone in the metabolism of Down syndrome

In Down syndrome (DS) in particular, the precise cellular mechanisms linking genotype to phenotype is not straightforward despite a clear mapping of the genetic cause. Metabolomic profiling might be more revealing in understanding molecular–cellular mechanisms of inborn errors of metabolism/syndromes than genomics alone and also result in new prenatal screening approaches. The urinary metabolome of 122 maternal urine from women with and without an aneuploid pregnancy (predominantly Down syndrome) were compared by both zwitterionic hydrophilic interaction chromatography (ZIC‐HILIC) and reversed‐phase liquid chromatography (RPLC) coupled to hybrid ion trap time of flight mass spectral analysis. ZIC‐HILIC mass spectrometry resolved 10‐fold more unique molecular ions than RPLC mass spectrometry, of which molecules corresponding to ions of m/z 114.07 and m/z 314.20 showed maternal urinary level changes that significantly coincided with the presence of a DS fetus. The ion of m/z 314.20 was identified as progesterone and m/z 114.07 as dihydrouracil. A metabolomics profiling‐based maternal urinary screening test modelled from this separation data would detect approximately 87 and 60.87% (using HILIC‐MS and RPLC‐MS, respectively) of all DS pregnancies between 9 and 23 weeks of gestation with no false positives. Copyright © 2014 John Wiley & Sons, Ltd.     

UPLC–QTOF/MS‐based metabolomics reveals the mechanism of chronic unpredictable mild stress‐induced hypertension in rats

Hypertension is a common chronic disease, and it is the strongest risk factor for cardiovascular disease. Recently, the number of patients with hypertension‐related complications has increased significantly, adding a heavy burden to the public health system. It is known that chronic stress plays an important role in the pathogenesis of cardiovascular diseases such as hypertension and stroke. However, the impact of hypertension on the dysfunctions induced by chronic stress remains poorly understood. In this study, using LC–MS‐based metabolomics, we established a chronic stress model to demonstrate the mechanisms of stress‐induced hypertension. We found that 30 metabolites in chronically stressed rats were changed; of these metabolites, seven had been upregulated, and 23 had been downregulated, including amino acids, phospholipids, carnitines and fatty acids, many of which are involved in amino acid metabolism, cell membrane injury, ATP supply and inflammation. These metabolites are engaged in dysregulated pathways and will provide a targeted approach to study the mechanism of stress‐induced hypertension.     

Evaluation of the combinative application of SDS and sodium deoxycholate to the LC–MS‐based shotgun analysis of membrane proteomes

SDS and sodium deoxycholate (SDC) as two representative detergents have been widely used in LC–MS/MS‐based shotgun analysis of membrane proteomes. However, some inherent disadvantages limit their applications such as interference with MS analysis or their weak ability to disrupt membranes. To address this, the combinative application of SDS and SDC was developed and evaluated in our study, which comprehensively used the strong ability of SDS to lyse membranes and solubilize hydrophobic membrane proteins, and the high efficiencies of an optimized acetone precipitation method and SDC in sample clean‐up, protein recovery, and redissolution and digestion of precipitated proteins. The comparative study using a rat‐liver‐membrane‐enriched sample showed that, compared with other three commonly used methods including the filter‐aided sample preparation strategy, the combinative method not only increased the identified number of total proteins, membrane proteins, and integral membrane proteins by an average of 19.8, 23.9, and 24.8%, respectively, but also led to the identification of the highest number of matching peptides. All these results demonstrate that the method yielded better recovery and reliability in the identification of the proteins especially highly hydrophobic integral membrane proteins than the other three methods, and thereby has more potential in shotgun membrane proteomics.     

UHPLC–Q‐TOF‐MS‐based metabolomics approach to compare the saponin compositions of Xueshuantong injection and Xuesaitong injection

Various traditional Chinese medicine preparations developed from Notoginseng total saponins, including Xueshuantong injection and Xuesaitong injection, are extensively used in China to treat cardiocerebrovascular diseases. However, the difference of their saponin compositions remains unknown. An ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry based metabolomics approach was developed to probe the saponin discrimination between Xueshuantong and Xuesaitong and the related factors by large sample analysis. A highly efficient chromatographic separation was achieved on an HSS T3 column within 20 min with the holistic metabolites information recorded in the negative MS^E mode. A six‐step data pretreatment procedure mainly based on Progenesis QI and mass defect filtering was established. Pattern recognition chemometrics was used to discover the potential saponin markers. The saponin composition of Wuzhou Xueshuantong showed distinct discrimination from the other products. Wuzhou Xueshuantong contains more abundant protopanaxatriol‐type noto‐R₁, Rg₁, Re, and protopanaxadiol‐type Rb₁, but less Rd and other low‐polarity protopanaxadiol‐type ginsenosides. These differences could not directly correlate to the use of different parts of Panax notoginseng , but possibly to the different preparation techniques employed by different manufacturers. These results are beneficial to the establishment of pharmacopoeia standards and the assessment of the efficacy and adverse drug reactions for these homologous products.     

Discovery of false identification using similarity difference in GC–MS‐based metabolomics

Compound identification is a critical process in metabolomics. The widely used approach for compound identification in gas chromatography–mass spectrometry‐based metabolomics is spectrum matching, in which the mass spectral similarity between an experimental mass spectrum and each mass spectrum in a reference library is calculated. While various similarity measures have been developed to improve the overall accuracy of compound identification, little attention has been paid to reducing the false discovery rate. We, therefore, develop an approach for controlling the false identification rate using the distribution of the difference between the first and second highest spectral similarity scores. We further propose a model‐based approach to achieving a desired true positive rate. The developed method is applied to the National Institute of Standards and Technology mass spectral library, and its performance is compared with that of the conventional approach that uses only the maximum spectral similarity score. The results show that the developed method achieves a significantly higher F1 score and positive predictive value than did the conventional approach. Copyright © 2014 John Wiley & Sons, Ltd.     

Rapid and robust confirmation and quantification of 11‐nor‐Δ9‐tetrahydrocannabinol‐9‐carboxylic acid (THC‐COOH) in urine by column switching LC‐MS‐MS analysis

A method for the rapid and robust confirmation of 11‐nor‐?9‐tetrahydrocannabinol‐9‐carboxylic acid (THCA) in urine involving basic hydrolysis with NaOH and direct injection of the hydrolysate in a column‐switching LC‐MS‐MS system was developed and validated. THCA‐d3 was used as internal standard. Detection was performed in negative‐ion mode by monitoring the transitions from the [M‐CO₂]‐ ion m/z 299.2→245.2 and and m/z 299.2→191.1 that were found to provide a better signal‐to‐noise ratio than the transition from the pseudomolecular ion at m/z 343. The high sensitivity of detection enabled the injection of a small volume (10 µl) of the NaOH hydrolysate which, together with the applied column switching system, proved to confer ruggedness to the method and to avoid the deterioration of the instrumental apparatus despite the large amount of inorganic ions in the hydrolysate. The LLOQ was established at 5 ng/ml, and the LLOD was calculated as 0.2 ng/ml (S/N =3). The method was submitted to thorough validation including evaluation of the calibration range (5–500 ng/ml), accuracy and precision, matrix effects, overall process efficiency, autosampler stability, carryover and cross‐talk, and 10‐times reduction of sample volume (0.1 ml). Proof of applicability was obtained by direct comparison with the reference GC‐MS method in use in the lab (the R² between the two methods was 0.9951). Copyright © 2012 John Wiley & Sons, Ltd.     

Solvent and Substituent Effects in the Solvolysis of 9‐Aryl‐9‐bromofluorenes

The solvolysis of eight 9‐aryl‐9‐bromofluorenes ( 6b～6i ) in a variety of solvents were studied. Correlation analysis using single‐parameter Grunwald‐Winstein equation (Eqn. 1) with different Y scales showed good linearity (R ≥ 0.98) for most cases if Y_xBnBr was employed. Linear relationships were observed from Hammett‐type analysis of logarithm of rate constants using Brown‐Okamoto σ⁺ constants (Eqn. 3), although inverse order of k(p‐CF₃)/k(m‐ CF₃) was realized in a number of cases. The ρ values were found to vary slightly with different solvent systems. Calculated atomic charge reveals the similarity between 9‐phenyl‐9‐fluorenyl cation ( 7 ) and triphenylmethyl cation ( 8 ). An extended charge delocalization throughout the fluorenyl ring led to the conclusion of the insignificance of antiaromaticity, which was in harmony with that obtained in previous studies. The variation of relative k_Br/k_Cl rate ratios was attributed to the electrophilic pull by solvents in solvolysis.     

Delipidation‐based solid‐phase extraction pretreatment technique for plasma broad‐coverage metabolomic profiling to reveal the potential pathogenesis of yeast‐induced fever in rats

During the process of metabolomics profiling by using ultra high performance liquid chromatography coupled with time‐of flight mass spectrometry, blood sample pretreatment is a crucial step for biomarker discovery. Herein, in order to prevent the potential loss of metabolites and ion suppression phenomena caused by the proteins and phospholipids contained in blood fluids, a delipidation‐based solid‐phase extraction pretreatment technique for plasma broad‐coverage metabolomic profiling was performed. This technique can be summarized as a single extraction, a single elution of solid‐phase extraction plate, followed by four times measuring with electrospray ionization in positive and negative ion mode, respectively. This approach significantly increased the number of features detected in plasma, and 1572 features in positive mode and 1352 features in negative mode were detected, respectively. Besides, the stability and repeatability of the approach were greatly improved. For these advantages, the approach was employed to elucidate the potential pathogenesis of yeast‐induced fever in rats. The biomarkers associated with the pathogenesis of fever were shown to be related to amino acids metabolism and lipid metabolism. The delipidation‐based solid‐phase extraction pretreatment approach can provide a useful tool to reveal the pathological mechanisms of such systemic pathological process.     

IEF pattern classification‐derived criteria for the identification of epoetin‐δ in urine

Epoetin‐δ (Dynepo? Shire Pharmaceuticals, Basing stoke, UK) is a synthetic form of erythropoietin (EPO) whose resemblance with endogenous EPO makes it hard to identify using the classical identification criteria. Urine samples collected from six healthy volunteers treated with epoetin‐δ injections and from a control population were immuno‐purified and analyzed with the usual IEF method. On the basis of the EPO profiles integration, a linear multivariate model was computed for discriminant analysis. For each sample, a pattern classification algorithm returned a bands distribution and intensity score (bands intensity score) saying how representative this sample is of one of the two classes, positive or negative. Effort profiles were also integrated in the model. The method yielded a good sensitivity versus specificity relation and was used to determine the detection window of the molecule following multiple injections. The bands intensity score, which can be generalized to epoetin‐α and epoetin‐β, is proposed as an alternative criterion and a supplementary evidence for the identification of EPO abuse.     

A survey of metabolic changes in potato leaves by NMR‐based metabolic profiling in relation to resistance to late blight disease under field conditions

Non‐targeted nuclear magnetic resonance (NMR)‐based metabolic profiling was applied to potato leaves to survey metabolic changes associated with late blight resistance under field conditions. Potato plants were grown in an experimental field, and the compound leaves with no visible symptoms were collected from 20 cultivars/lines at two sampling time points: (i) the time of initial presentation of symptoms in susceptible cultivars and (ii) 12 days before this initiation. ¹H NMR spectra of the foliar metabolites soluble in deuterium oxide‐ or methanol‐d₄‐based buffers were measured and used for multivariate analysis. Principal component analysis for six cultivars at symptom initiation showed a class separation corresponding to their levels of late blight resistance. This separation was primarily explained by higher levels of malic acid, methanol, and rutin and a lower level of sucrose in the resistant cultivars than in the susceptible ones. Partial least squares regression revealed that the levels of these metabolites were strongly associated with the disease severity measured in this study under field conditions. These associations were observed only for the leaves harvested at the symptom initiation stage, but not for those collected 12 days beforehand. Subsequently, a simple, alternative enzymatic assay for l ‐malic acid was used to estimate late blight resistance, as a model for applying the potential metabolic marker obtained. This study demonstrated the potential of metabolomics for field‐grown plants in combination with targeted methods for quantifying marker levels, moving towards marker‐assisted screening of new cultivars with durable late blight resistance. Copyright © 2016 John Wiley & Sons, Ltd.     

Thioxanthone–anthracene‐9‐carboxylic acid as radical photoinitiator in the presence of atmospheric air

Thioxanthone–anthracene‐9‐carboxylic acid (TX‐ANCA) namely 14‐oxo‐14H‐naphthol [2,3‐b]thioxanten‐12‐carboxylic acid, is synthesized and characterized as part of our continuing interest for syntheses of polyaromatic initiators. Photoinitiator, TX‐ANCA have good absorption properties in the UV and visible region of the electromagnetic spectrum (ɛ₃₇₀: 9080 M⁻¹cm⁻¹, ɛ₄₃₀: 6151 M⁻¹ cm⁻¹). The fluorescence quantum yield is calculated as 0.1 which is slightly higher than of the parent thioxanthone compound (φ_f: 0.07). The phosphorescence lifetime is found to be 39 ms. The possible initiating mechanism of TX‐ANCA is based on photoexcitation of TX‐ANCA and quenching of triplet excited states of TX‐ANCA by molecular oxygen generates singlet oxygen. Singlet oxygen reacts with the anthracene moiety of TX‐ANCA possibly forms an endoperoxide. The endoperoxides undergoes photochemical or thermal decomposition to form radicals which are able to initiate free radical polymerization. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 1878–1883     

Nontypical iodine–halogen bonds in the crystal structure of (3E)‐8‐chloro‐3‐iodomethylidene‐2,3‐dihydro‐1,4‐oxazino[2,3,4‐ij]quinolin‐4‐ium triiodide

Two kinds of iodine–iodine halogen bonds are the focus of our attention in the crystal structure of the title salt, C₁₂H₈ClINO⁺·I₃⁻, described by X‐ray diffraction. The first kind is a halogen bond, reinforced by charges, between the I atom of the heterocyclic cation and the triiodide anion. The second kind is the rare case of a halogen bond between the terminal atoms of neighbouring triiodide anions. The influence of relatively weakly bound iodine inside an asymmetric triiodide anion on the thermal and Raman spectroscopic properties has been demonstrated.     

A metabolomic strategy to screen the prototype components and metabolites of Qingkailing injection in rat urine by high‐performance liquid chromatography with tandem mass spectrometry

Xenobiotic metabolome identification of Chinese herbal formula in biological systems is a very challenging task. Qingkailing injection is a typical Chinese herbal injection, which is wildly used clinically in China. However, the holistic metabolic fate of the ingredient from Qingkailing injection remains unclear. In this work, a metabolomic strategy for comprehensively elucidating Qingkailing injection derived prototype components and metabolites in rat urine conducted by hybrid linear ion trap high‐resolution mass spectrometry was developed. High‐performance liquid chromatography coupled with hybrid linear ion trap high‐resolution mass spectrometry was developed to obtain the urine profiling between the control group and Qingkailing injection treated group. Orthogonal partial least squares discriminate analysis was applied to distinguish the exogenous and the endogenous. In the S‐plot, 37 xenobiotics derived from Qingkailing injection were found in urine, including 18 prototype compounds and 19 metabolites. The characterization of the prototype components and metabolites in rat's urine provided essential data for further pharmacological studies of Qingkailing injection. Our results indicated that the metabolomic approach was an effective tool to discover, screen, and analyze the multiple prototype components and their metabolites from complicated traditional Chinese preparations in vivo.     

Copolymerization of propylene with higher α‐olefins in the presence of the syndiospecific catalyst i‐Pr(Cp)(9‐Flu)ZrCl2/MAO

The catalyst system i‐Pr(Cp)(9‐Flu)ZrCl₂/methylaluminoxane was used for the synthesis of random syndiotactic copolymers of propylene with 1‐hexene, 1‐dodecene, and 1‐octadecene as comonomers. An investigation of the microstructure by ¹³C NMR spectroscopy revealed that the stereoregularity of the copolymers decreased because of an increase in skipped insertions in the presence of the higher 1‐olefin. The melting temperature of the copolymers, as measured by differential scanning calorimetry (DSC), decreased linearly with increasing comonomer content independently of the comonomer nature. During the DSC heating cycle, an exothermic peak indicating a crystallization process was observed. The decrease in the crystallization temperature with higher 1‐olefin content, measured by crystallization analysis fractionation, indicated a small but significant dependence on the nature of the comonomer. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 40: 128–140, 2002     

Ionic‐liquid‐based dispersive liquid–liquid microextraction coupled with high‐performance liquid chromatography for the forensic determination of methamphetamine in human urine

Determination of methamphetamine in forensic laboratories is a major issue due to its health and social harm. In this work, a simple, sensitive, and environmentally friendly method based on ionic liquid dispersive liquid–liquid microextraction combined with high‐performance liquid chromatography was established for the analysis of methamphetamine in human urine. 1‐Octyl‐3‐methylimidazolium hexafluorophosphate with the help of disperser solvent methanol was selected as the microextraction solvent in this process. Various parameters affecting the extraction efficiency of methamphetamine were investigated systemically, including extraction solvent and its volume, disperser solvent and its volume, sample pH, extraction temperature, and centrifugal time. Under the optimized conditions, a good linearity was obtained in the concentration range of 10–1000 ng/mL with determination coefficient >0.99. The limit of detection calculated at a signal‐to‐noise ratio of 3 was 1.7 ng/mL and the relative standard deviations for six replicate experiments at three different concentration levels of 100, 500, and 1000 ng/mL were 6.4, 4.5, and 4.7%, respectively. Meanwhile, up to 220‐fold enrichment factor of methamphetamine and acceptable extraction recovery (>80.0%) could be achieved. Furthermore, this method has been successfully employed for the sensitive detection of a urine sample from a suspected drug abuser.     

Investigating the robustness and extraction performance of a matrix‐compatible solid‐phase microextraction coating in human urine and its application to assess 2–6‐ring polycyclic aromatic hydrocarbons using GC–MS/MS

In this work, a polydimethylsiloxane/divinylbenzene fiber overcoated with a layer of polydimethylsiloxane was evaluated as analytical sampling tool for the first time in human urine. Urinary polycyclic aromatic hydrocarbons with 2–6 aromatic rings were considered as target compounds. The analyte uptake in kinetic and thermodynamic regime was evaluated and compared to the performances of polydimethylsiloxane/divinylbenzene and polydimethylsiloxane fibers. The assessment of the robustness and endurance of the overcoated fiber was carried out by direct immersion solid‐phase microextraction in undiluted urine performing up to 120 consecutive extractions. The overcoated fiber was then used to develop a fast and easy direct immersion solid‐phase microextraction with gas chromatography and triple quadrupole mass spectrometry protocol for the quantification of the target polycyclic aromatic hydrocarbons. The attained values of accuracy and precision were 75–114% and 2–19%, respectively, while the limits of quantification ranged between 0.05 and 1 ng/L. The proposed protocol was applied to the screening of urine samples collected from smoking and nonsmoking volunteers. The successful results obtained by using the overcoated fiber create not only new alternatives for polycyclic aromatic hydrocarbon exposure assessment but also new perspectives for the application of direct immersion solid‐phase microextraction to the analysis of bioclinical matrixes.     

Ultra‐rapid non‐equilibrium solid‐phase microextraction at elevated temperatures and direct coupling to mass spectrometry for the analysis of lidocaine in urine

Solid‐phase microextraction (SPME) has been directly coupled to an ion‐trap mass spectrometer (MS) for the determination of the model compound lidocaine in urine, hereby applying MS/MS [fragmentation of [M + H]⁺ (m/z 235) to a fragment with m/z 86]. The throughput of samples has been increased using non‐equilibrium SPME with polydimethylsiloxane (PDMS) fibers. The effect of temperature on the sorption and the desorption was studied. Elevated temperatures during sorption (65°C) and desorption (55°C) had a considerable influence on the speed of the extraction. The desorption was carried out with a home‐made desorption chamber allowing thermostating. Only 1 min sorption and 1 min desorption were performed, after which MS detection took place, resulting in a total analysis time of 3 min. Detection limits below 1 ng/mL could be obtained despite yields of only 2.1 and 1.5% for a 100‐ and a 30‐μm PDMS‐coated fiber, respectively. Furthermore, the determination of lidocaine in urine had acceptable reproducibilities, i.e., relative standard deviations (RSDs) below 10%. A limit of quantitation (RSD < 15%) of about 1 ng/mL was obtained. No extra wash step of the extraction fiber was required after desorption if a 30‐μm coating was used, whereas not all the analyte was desorbed from the 100‐μm coating in a single desorption. Therefore, the SPME‐MS/MS system with a 30‐μm PDMS‐coated fiber for rapid non‐equilibrium SPME at elevated temperatures has interesting potential for high‐throughput analysis of biological samples.