Early prediction of wheat quality: analysis during grain development using mass spectrometry and multivariate data analysis

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and multivariate data analysis have been used for the determination of wheat quality at different stages of grain development. Wheat varieties with one of two different end-use qualities (i.e. suitable or not suitable for bread-making purposes) were investigated. The samples were collected from grains from 15 until 45 days post-anthesis (dpa). Gluten proteins from wheat grains were extracted and subsequently analysed by mass spectrometry. Discrimination partial least-squares regression and soft independent modelling of class analogy were used to determine the quality of new and unknown wheat samples. With these methods, we were able to predict correctly the end-use qualities at every stage investigated. This new fast technique, based on the rapidity of mass spectrometry combined with the objectivity of multivariate data analysis, offers a method that can replace the traditional rather time-consuming ones such as gel electrophoresis. This study focused on the determination of wheat quality at 15 dpa, when the grain is due for harvest 1 month later.     

Orthogonal projection (OP) technique applied to pattern recognition of fingerprints of the herbal medicine houttuynia cordata Thunb. and its final injection products

It is a crucial issue to determine the origins of herbal medicinal materials and identify the quality grades and fakes of their final products collected from different pharmaceutical corporations. Pattern recognition technique may assist the manufacturers to achieve this purpose and effectively control the quality of their products. In this work, a widely used method in chemometrics, orthogonal projection (OP) technique, was applied to discrimination analysis and identification of fingerprints of the herbal medicine houttuynia cordata Thunb. (HCT) and its final injection products. The advantages of the OP technique are clearly shown after comparing with the conventional methods such as principal component analysis (PCA), Mahalanobis distance (MD), and similarity comparison method (SCM). Three different sources of medicinal material HCT and its final injection products from six different manufacturers were studied under 'sixfold', 'threefold' and 'threefold-bis' cross-validation procedures. The good performance of the proposed method in determination and identification of unknown samples shows it could be a powerful tool for quality control in herbal medicine production and other related research fields.     

Microbial growth on pall rings

Biogas is upgraded using an absorption with water-wash technique by 11 of a total of 14 upgrading plants in Sweden. However, problems with microbial growth on the pall rings in the absorption column, and in one case in the desorption column, have a negative impact on the upgrading of raw gas to vehicle gas. Five of the nine biogas plants studied here have experienced problems with microbial growth. The objectives of this study were to identify such microbial growth and to determine possible factors for its control, in order to provide recommendations for process management. A questionnaire was sent out and visits were made to the upgrading plants to collect information about the upgrading process. Phospholipid fatty acid (PLFA) analysis was performed to determine microbial biomass and community structure in samples from four upgrading plants. In samples from two of the plants, methane-oxidizing bacteria (type I methanotrophs) were indicated, while samples from one of the other plants showed biomarkers indicating actinomycetes. Factors affecting development of microbial growth were found to be water quality and the pH and temperature of the process water. Plants that used wastewater in the upgrading process experienced far more problems than those using clean water of drinking quality.     

The Merits of the High-temperature Combustion Method for Determining the Amount of Natural Organic Carbon in Surface Freshwater Samples

The merits of the method for the quantitative analysis of carbon of natural organic matter (NOM) in aquatic freshwater samples, using the high-temperature catalytic combustion technique, have to our knowledge not previously been documented. Although a large intercalibration study concerning marine NOM have documented and improved the analytical merits of this technique in marine chemistry, these results cannot directly be adapted to freshwater analysis. This article presents the findings from an intercalibration on the determination of carbon in freshwater NOM, between 25 laboratories participated in using the high-temperature catalytic combustion technique. The laboratories analysed the amount of total- and dissolved organic carbon (i.e. TOC and DOC, respectively) on a set of 10 samples. The samples consisted of fresh surface water, reverse osmosis (RO) isolates of dissolved NOM from natural freshwater locations, as well as synthetic standards. General merits (i.e. accuracy, precision etc.) of the analytical method are presented and problems with detection limits, high blank values, contamination from filters, experience in the use of RO-isolates and analysis of the more refractory (i.e. not readily oxidizable) NOM are discussed. The focus of the study is on the analytical merits achieved on the natural samples compared to the commonly used and possibly readily oxidizable standard material (i.e. potassium hydrogen phthalate). It is demonstrated that the method's merits generated using the readily oxidizable potassium hydrogen phthalate are too optimistic when applied to the analysis of natural aquatic samples.     

Rapid monitoring and sensitive determination of DDT and its metabolites in water sample using solid-phase extraction followed by ion mobility spectrometry

Dichlorodiphenyl trichloroethane (DDT) as an organochlorine compound has been globally used as a pesticide for controlling soil-dwelling insects and treating diseases such as malaria and typhus. The degradation products of DDT and its metabolites have also negative effects on the environment. The present study has investigated the determination of DDT and its metabolites in water sample using ion mobility spectrometry (IMS) as a rapid and sensitive detection technique. For this purpose, DDT and its metabolites were extracted using reverse phase solid-phase extraction (SPE) from water samples. The samples were then recovered by eluting with methanol and finally, quantified using the corona discharge IMS technique. Injection and oven temperatures and the effect of dopant were optimized as experimental parameters influencing both detection and determination efficiencies. Degradation of DDT in IMS drift tube was studied and reduced mobility values of DDT and its metabolites were calculated. The developed method was validated using water sample to obtain good results for the determination of DDT at low levels (1 ng ml^?1) while spiked recoveries were obtained to be between 95.0–96.7%. The proposed method based on IMS proved to be a simple, inexpensive, rapid and sensitive procedure for the fast monitoring and determination of DDT and its main metabolites in water sample.     

Determination of Metabolites from Scutellaria baicalensis by GC/MS and 1H NMR

The quality of medicinal herbs and their products is steadily becoming important in tandem with growing interest in complementary alternative medicine for treatment of diseases. Chemical assays with bioactive secondary compounds are usually used for chemical standardization in herbs for quality control purpose. In this study, an analytical platform comprising of a GC-MS technique with an unsupervised multivariate analysis and a complementary one-dimensional ¹H NMR technique was used to obtain the primary metabolite profiling of Scutellaria baicalensis obtained from different medical halls in Singapore. The key primary metabolites such as sucrose, proline, phenylalanine, fructose, and butanedioic acid for the biosynthesis of bioactive secondary metabolites in this medicinal herb were successfully characterized by the combination of the chromatography and spectroscopy techniques. Their results suggested that these compounds could serve as markers for quality control of the herb. The principal component analyses of the GC-MS data reliably discriminated between the various Scutellaria samples indicating that the developed platform was comprehensive and was applicable to assess the quality of other medicinal herbs.     

Detection of volatile metabolites of <Emphasis Type="Italic">Escherichia coli</Emphasis> by multi capillary column coupled ion mobility spectrometry

Detection and immediate quantification of microbial metabolic activities is of high interest in fields as diverse as biotechnology and infection biology. Interestingly, the most direct signals of microbial metabolism, the evolution of volatile metabolites, is largely ignored in the literature, and rather, metabolite concentrations in the microbial surrounding or even disruptive methods for intracellular metabolite measurements (i.e., metabolome analysis) are favored. Here, the development of a multi capillary column coupled ion mobility spectrometer (MCC-IMS) was described for the detection of volatile organic compounds from microbes and the MCC-IMS was used for characterization of metabolic activity of growing Escherichia coli. The MCC-IMS chromatogram of the microbial culture off-gas of the acetone-producing E. coli strain BL21 pLB4 revealed four analytes that positively correlated with growth, which were identified as ethanol, propanone (acetone), heptan-2-one, and nonan-2-one. The occurrence of these analytes was cross-validated by solid-phase micro-extraction coupled with gas chromatography mass spectrometry analysis. With this information in hand, the dynamic relationship between the E. coli biomass concentration and the metabolite concentrations in the headspace was measured. The results suggest that the metabolic pathways of heptan-2-one and nonan-2-one synthesis are regulated independent of each other. It is shown that the MCC-IMS in-line off-gas analysis is a simple method for real-time detection of microbial metabolic activity and discussed its potential for application in metabolic engineering, bioprocess control, and health care.     

LC-ESI-MS分析两性霉素B降解产物

两性霉素B(amphotericinB)是一种常用的抗真菌抗生素,属于多烯大环内酯类化合物,化学性质不稳定,在生产和储存过程中容易发生多种降解反应,产生无活性甚至有毒性的降解物,直接影响产品质量和临床用药的安全,因此,研究该抗生素降解物和降解途径,对防止产品降解、保证产品质量非常必要。已有不少有关两性霉素B各种剂型稳定性的研究报道,ThomaK等研究报道了多烯类抗生素光稳定性结果,然而有关两性霉素B在酸、碱、加热、氧化和光照等条件下降解途径研究未见报道。由于直接分离鉴定各降解产物比较困难,本实验采用LC-MS在线分析手段,直接分析检测降解产物。实验表明,通过检测各种加速实验样品的降解物,可以确立降解途径和降解物,本文对控制降解反应发生、提高产品质量以及稳定性研究具有重要意义。     

Exemplar application of multi-capillary column ion mobility spectrometry for biological and medical purpose

In recent years, ion mobility spectrometry is increasingly in demand for new applications especially on biological samples (cells, bacteria, fungi), in medicine (diagnosis, therapy and medication control e.g. from breath analyses), for food quality control, safety monitoring and characterisation or process control in chemical and pharmaceutical industry. For this purpose instruments based on gas phase separation of ions in weak electric fields were developed at ISAS–Institute for Analytical Sciences, focussing on the particular challenges such as humid and rather complex samples, specific sampling procedures adapted to the application, fast pre-separation techniques like multi-capillary columns and suitable data processing including data bases for relevant analytes and automatic characterisation of IMS-chromatograms. Feasibility studies were carried out successfully for biological and medical purpose at ISAS, including the detection of bacteria, fungi and metabolites of cells and in human breath. For all those samples characteristic pattern of analytes were found and could be used for the identification of cell lines, fungi and bacteria as well as of numerous diseases. Furthermore, the quantification of those analytes could be used to obtain information about the state of the process or person (e.g. growth of cultures, development of diseases, level of medication, grade of cancer). Those examples shall demonstrate the potential of ion mobility spectrometry for the selected applications. However, a general and reliable data bases of reference analytes is required in the near future to enable an exploitation of the metabolic pathways and to confirm the relevance of the detected signals for the investigated topic.     

Preprocessing and exploratory analysis of chromatographic profiles of plant extracts

The characterization of herbal extracts to compare samples from different origin is important for robust production and quality control strategies. This characterization is now mainly performed by analysis of selected marker compounds. Metabolic fingerprinting of full metabolite profiles of plant extracts aims at a more rapid and thorough screening or classification of plant material. We will show that HPLC is an appropriate technique for metabolic fingerprinting of secondary metabolites, given that adequate preprocessing of raw profiles is performed. Additional variation, which results from sample preparation and changing measurement conditions, usually obscures the information of interest in these raw profiles. This paper illustrates the importance of preprocessing of chromatographic fingerprinting data. Different alignment methods are discussed as well as the influence of normalization. Weighted principal component analysis is introduced as a valuable alternative to autoscaling of data. LC-UV data on Willow (Salix sp.) extracts is used to evaluate these preprocessing methods and their influence on exploratory data analysis.     

Improvement of quality control of speciation analysis using hyphenated techniques A decade of progress within the European Community

The awareness of a need for an improved control of environmental contamination levels has led to the development of new hyphenated techniques for the determination of a wide variety of chemical species (e.g., organotins, methyl-mercury, alkyl-lead compounds etc.). These techniques generally involve many analytical steps such as extraction, derivatisation, separation and detection which have to be carried out in such a way that the speciation is not changed during the abalytical process. The need for evaluating the method's performance has led the BCR programme of the European Commission (now Standards, Measurements and Testing programme) to conduct series of interlaboratory studies during the last decade. These projects followed a step-by-step approach for the evaluation of different steps of the analytical methods used, e.g., simple solutions to test the detection, cleaned extract to evaluate the separation, spiked samples to test the extraction and natural samples to evaluate the whole analytical procedures. These collaborative projects allowed most of the sources of errors related to either a technique or a laboratory to be detected and removed. This paper gives an account of discussions of possible errors occurring in speciation analysis and presents examples of technical scrutiny of hyphenated techniques using chromatography as applied to the determination of tributyltin, methyl-mercury and trimethyllead.     

Determination of Arsenic and Mercury in Various Environmental Matrices by Chemical Neutron Activation Analysis

Chemical neutron activation analysis was developed for determining trace amounts of arsenic and mercury in a variety of environmental matrices, including water, sediment, rock, plants, animal organs, etc: The adsorption procedure via magnesium oxide as the agent was applied to preconcentrate arsenic from the digested environmental matrices where arsenic in the form of As(V) ion could be highly efficiently adsorbed by hydrous magnesium oxide. On the other hand, the extraction procedure via lead diethyldithiocarbamate as the agent was applied to preconcentrate mercury from the digested environmental matrices where mercury in the form of Hg(II) ion could be highly efficiently extracted into the solution of lead diethyldithiocarbamte in dichloromethane. Both of the preconcentrated materials prepared ultimately in the solid states, i.e., arsenic in magnesium oxide and mercury in lead diethyldithiocarbamte were taken to be neutron irradiated. The γ spectra of the preconcentrated samples irradiated generally showed clear peaks of the product radionuclides from arsenic or from mercury by the different separation procedures. The possible interfering elements such as Na, Br, etc., were prominently minimized in respect of most of the preconcentrated samples. The reliability and accuracy of the proposed analytical methods for detecting arsenic and mercury can be confirmed by the assay of commercial standard reference materials including sediment, rock, plants, and animal organs.     

非靶标代谢组研究温度对黄曲霉菌代谢的影响

采用基于超高效液相色谱-高分辨质谱联用的非靶标代谢组学方法来研究温度对黄曲霉菌生理代谢的影响,使用交互偏最小二乘判别分析(Ortho PLS-DA)等化学计量学方法对代谢组数据进行多元统计分析,使用二级质谱信息和谱库检索定性黄曲霉菌代谢特征信息。使用内标结合混合质控样品的方法对非靶标代谢组方法进行质量控制。将该方法应用于研究温度对黄曲霉菌代谢组的影响,发现不同温度下有3 593个(T检验p0.01)差异表达代谢特征,筛选出20个候选差异代谢物。研究结果表明,温度显著影响三羧酸循环、脂肪酸、苯丙氨酸、色氨酸、络氨酸等生物合成路径,并调控黄曲霉毒素、黄匹阿尼酸、曲酸等次生代谢物生物合成路径酶活性。研究发现曲酸和黄曲霉毒素前体化合物与黄曲霉毒素的累积变化规律相似,可作为候选靶标进行验证。该研究为开展我国黄曲霉毒素风险评估和分子预警研究提供了新的路径和方法。     

The chemical ecology of crucifers and their fungal pathogens: boosting plant defenses and inhibiting pathogen invasion

Fungal plant diseases can cause very substantial yield losses in crucifer crops such as rapeseed and canola, or vegetables such as cabbage and broccoli. To devise sustainable methods to prevent and deter crucifer pathogens, the chemical interaction between crucifers and their fungi is under intense investigation. Crucifers produce complex blends of secondary metabolites with diverse ecological roles that include protection against microbial pathogens and other pests. The secondary metabolites involved in crucifer defense, namely phytoalexins and phytoanticipins, and their metabolism by fungal pathogens indicate that some fungi produce different enzymes to detoxify these metabolites and that some fungal detoxifying enzymes are rather specific. Chemical synthesis and screening of phytoalexin analogue libraries using cultures of fungal pathogens, as well as protein extracts, have shown that such detoxification reactions can be inhibited and that some inhibitors are strongly antifungal. Overall results of current work show the feasibility of using selective inhibitors of fungal detoxifying enzymes, i.e., paldoxins, to protect plants by boosting their chemical defenses.     

Real-time 2D separation by LC × differential ion mobility hyphenated to mass spectrometry

The liquid chromatography–mass spectrometry (LC-MS) analysis of complex samples such as biological fluid extracts is widespread when searching for new biomarkers as in metabolomics. The success of this hyphenation resides in the orthogonality of both separation techniques. However, there are frequent cases where compounds are co-eluting and the resolving power of mass spectrometry (MS) is not sufficient (e.g., isobaric compounds and interfering isotopic clusters). Different strategies are discussed to solve these cases and a mixture of eight compounds (i.e., bromazepam, chlorprothixene, clonapzepam, fendiline, flusilazol, oxfendazole, oxycodone, and pamaquine) with identical nominal mass (i.e., m/z 316) is taken to illustrate them. Among the different approaches, high-resolution mass spectrometry or liquid chromatography (i.e., UHPLC) can easily separate these compounds. Another technique, mostly used with low resolving power MS analyzers, is differential ion mobility spectrometry (DMS), where analytes are gas-phase separated according to their size-to-charge ratio. Detailed investigations of the addition of different polar modifiers (i.e., methanol, ethanol, and isopropanol) into the transport gas (nitrogen) to enhance the peak capacity of the technique were carried out. Finally, a complex urine sample fortified with 36 compounds of various chemical properties was analyzed by real-time 2D separation LC×DMS-MS(/MS). The addition of this orthogonal gas-phase separation technique in the LC-MS(/MS) hyphenation greatly improved data quality by resolving composite MS/MS spectra, which is mandatory in metabolomics when performing database generation and search.     

Semi-targeted analysis of metabolites using capillary-flow ion chromatography coupled to high-resolution mass spectrometry

This work describes a novel application of capillary-flow ion chromatography mass spectrometry for metabolomic analysis, and comparison of the technique to octadecyl silica and hydrophilic interaction chromatography (HILIC)-based mass spectrometry. While liquid chromatography/mass spectrometry (LC/MS) is rapidly becoming the standard technique for metabolomic analysis, metabolomic samples are extremely heterogeneous, leading to a requirement for multiple methods of analysis and separation techniques to perform a 'global' metabolomic analysis. While C18 is suitable for hydrophobic metabolites and has been used extensively in pharmaceutical drug metabolism studies, HILIC is, in general, efficient at separating polar metabolites. Phosphorylated species and organic acids are challenging to analyse and effectively quantitate on both systems. There is therefore a requirement for an MS-compatible analytical technique that can separate negatively charged compounds, such as ion-exchange chromatography. Evaluation of capillary flow ion chromatography with electrolytic suppression was performed on a library of metabolite standards and was shown to effectively separate organic acids and sugar di- and tri-phosphates. Limits of detection for these compounds range from 0.01 to 100 pmol on-column. Application of capillary ion chromatography to a comparative analysis of energy metabolism in procyclic forms of the parasitic protozoan Trypanosoma brucei where cells were grown on glucose or proline as a carbon source was demonstrated to be more effective than HILIC for detection of the organic acids that comprise glucose central metabolism and the tricarboxylic acid (TCA) cycle.     

Green Bioanalytical Applications of Graphene Oxide for the Extraction of Small Organic Molecules

Bioanalysis is the scientific field of the quantitative determination of xenobiotics (e.g., drugs and their metabolites) and biotics (e.g., macromolecules) in biological matrices. The most common samples in bioanalysis include blood (i.e., serum, plasma and whole blood) and urine. However, the analysis of alternative biosamples, such as hair and nails are gaining more and more attention. The main limitations for the determination of small organic compounds in biological samples is their low concentration in these matrices, in combination with the sample complexity. Therefore, a sample preparation/analyte preconcentration step is typically required. Currently, the development of novel microextraction and miniaturized extraction techniques, as well as novel adsorbents for the analysis of biosamples, in compliance with the requirements of Green Analytical Chemistry, is in the forefront of research in analytical chemistry. Graphene oxide (GO) is undoubtedly a powerful adsorbent for sample preparation that has been successfully coupled with a plethora of green extraction techniques. GO is composed of carbon atoms in a sp² single-atom layer of a hybrid connection, and it exhibits high surface area, as well as good mechanical and thermal stability. In this review, we aim to discuss the applications of GO and functionalized GO derivatives in microextraction and miniaturized extraction techniques for the determination of small organic molecules in biological samples.     

Determination of diphenamide, napropamide and metolachlor in tobacco by gel permeation chromatographic clean-up and high performance liquid chromatography

Diphenamide, napropamide and metolachlor (FIG. 1) are selective, pre-emergence arylamide herbicides used to control the growth of annual grasses and broadleaf weeds in a variety of fields, e.g. fruit trees, nuts, corns, green crops, etc. They possess high activity and moderate toxicity. For food and environment safety, the detailed investigations on their residues and metabolism are very important. Diphenamide, napropamide and metolachlor in the pesticide products, serum, urine, soil, environmental water, fruits and wine have been widely analyzed by ELISA, fluorescence, phosphorescence, capillary electrophoresis, high performance liquid chromatography (HPLC), gas chromatography(GC) and GC mass spectrometry (GC-MS). However, to our knowledge, simultaneous residue analysis of diphenamide, napropamide and metolachlor in tobacco samples has not been extensively documented. Tobacco is greatly consumed by smokers throughout the world. The pesticide residue in tobaccos might be potentially harmful to smokers' health. With this in mind the residue determination and control of diphenamide, napropamide and metolachlor in the tobacco leaves are very important for tobacco products and consumers. For these three herbicides, the tolerable maximum residue limits (MRLs) have been limited ranging from 0.05 (for tobacco products) to 5 mg/kg (for tobacco leaves) in different European countries. For the complex tobacco samples, the GC and HPLC with UV detection suffer from matrix interference making quantification and identification of these herbicides difficult. In such cases the removal of the matrix effects and identification of the target compounds are of great importance. The present work reports the extraction and clean up procedures, as well as, the chromatographic conditions developed for the simultaneous determination of diphenamide, napropamide and metolachlor residues in the fluecured tobacco leaves, from the different sources using HPLC-UV method.     

MALDI-ToF mass spectrometry coupled with multivariate pattern recognition analysis for the rapid biomarker profiling of Escherichia coli in different growth phases

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) has been exploited extensively in the field of microbiology for the characterisation of bacterial species, the detection of biomarkers for early disease diagnosis and bacterial identification. Here, the multivariate data analysis technique of partial least squares-discriminant analysis (PLS-DA) was applied to ‘intact cell’ MALDI-ToF MS data obtained from Escherichia coli cell samples to determine if such an approach could be used to distinguish between, and characterise, different growth phases. PLS-DA is a technique that has the potential to extract systematic variation from large and noisy data sets by identifying a lower-dimensional subspace that contains latent information. The application of PLS-DA to the MALDI-ToF data obtained from cells at different stages of growth resulted in the successful classification of the samples according to the growth phase of the bacteria cultures. A further outcome of the analysis was that it was possible to identify the mass-to-charge (m/z) ratio peaks or ion signals that contributed to the classification of the samples. The Swiss-Prot/TrEMBL database and primary literature were then used to provisionally assign a small number of these m/z ion signals to proteins, and these tentative assignments revealed that the major contributors from the exponential phase were ribosomal proteins. Additional assignments were possible for the stationary phase and the decline phase cultures where the proteins identified were consistent with previously observed biological interpretation. In summary, the results show that MALDI-ToF MS, PLS-DA and a protein database search can be used in combination to discriminate between ‘intact cell’ E. coli cell samples in different growth phases and thus could potentially be used as a tool in process development in the bioprocessing industry to enhance cell growth and cell engineering strategies.