Gas chromatography-mass spectrometric method for metabolic profiling of tobacco leaves

A gas chromatography-mass spectrometric method was developed for profiling of tobacco leaves. The differentiation among tobacco leaves planted in two different regions was investigated. Prior to analysis, the extraction solvent formulation was optimized and a combination of water, methanol and acetonitrile with a volume ratio of 3:1:1 was found to be optimal. The reproducibility of the method was satisfactory. Kendall tau-b rank correlation coefficients were equal to 1 (p<0.05) for 82% of the resolved peaks (up to 95% of the overall peak areas), indicating the good response correlation. Forty-four compounds including 9 saccharides, 9 alcohols, 9 amino acids, 16 organic acids and phosphoric acid were identified based on standard compounds. The method was successfully applied for profiling of tobacco leaves from Zimbabwe and Yunnan of China. Our result revealed that levels of saccharides and their derivatives including xylose, ribose, fructose, glucose, turanose, xylitol and glyceric acid were more abundant while sucrose, glucitol and D-gluconic acid were less abundant in tobacco leaves from Yunnan as compared to those from Zimbabwe. Amino acids such as L-alanine, L-tyrosine and L-threonine were found to be richer in Zimbabwe tobacco than in Yunnan tobacco.     

Low-molecular weight protein profiling of genetically modified maize using fast liquid chromatography electrospray ionization and time-of-flight mass spectrometry

In this work, the use of liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC-TOFMS) has been evaluated for the profiling of relatively low-molecular weight protein species in both genetically modified (GM) and non-GM maize. The proposed approach consisted of a straightforward sample fractionation with different water and ethanol-based buffer solutions followed by separation and detection of the protein species using liquid chromatography with a small particle size (1.8 μm) C(18) column and electrospray-time-of-flight mass spectrometry detection in the positive ionization mode. The fractionation of maize reference material containing different content of transgenic material (from 0 to 5% GM) led to five different fractions (albumins, globulins, zeins, zein-like glutelins, and glutelins), all of them containing different protein species (from 2 to 52 different species in each fraction). Some relevant differences in the quantity and types of protein species were observed in the different fractions of the reference material (with different GM contents) tested, thus revealing the potential use of the proposed approach for fast protein profiling and to detect tentative GMO markers in maize.     

Lipid profiling of cyanobacteria Synechococcus sp. PCC 7002 using two‐dimensional liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Glycerolipid is a main component of membranes in oxygenic photosynthetic organisms. Up to now, the majority of publication in this area has focused on the physiological functions of glycerolipids and lipoprotein complexes in photosynthesis, but the study on the separation and identification of glycerolipids in thylakoid membrane in cyanobacteria is relatively rare. Here we report a new method to separate and identify five photosynthetic glycerolipid classes, including monoglucosyl diacylglycerol, monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol, in cyanobacteria Synechococcus sp. PCC 7002 by two‐dimensional (normal‐ and reversed‐phase) liquid chromatography online coupled to quadrupole time‐of‐flight mass spectrometry. Over twice as many lipid species were detected by our method compared to the previously reported methods. Ten new odd‐chain fatty acid glycerolipids were discovered for the first time. Moreover, complete separation of isomers of monogalactosyl diacylglycerol and monoglucosyl diacylglycerol was achieved. According to the tandem mass spectrometry results, we found that the head group of monoglucosyl diacylglycerols was not as stable as that of monogalactosyl diacylglycerols, which might explain why the organism chose monogalactosyl diacylglycerols and digalactosyl diacylglycerols instead of monoglucosyl diacylglycerols as the main content of the photosynthetic membranes in the history of evolution. This work will benefit further research on the physiological function of glycerolipids.     

Systematic screening and characterization of glycosides in tobacco leaves by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry using neutral loss scan and product ion scan

Glycosides in tobacco leaves are highly important aromatic precursors. It is necessary to reveal glycosides in tobacco leaves to improve tobacco planting and processing. This study describes a method for the systematic screening of glycosides in tobacco leaves by liquid chromatography with tandem mass spectrometry. Although glycosides contain numerous aglycones, the number of glycans is limited. Based on a screening table of glycans designed for neutral loss scan, glycosides with different aglycones were systematically screened out. Then, the MS² fragment spectra of scanned glycosides were further obtained using product ion scan. By comparison with the spectra in online tandem mass spectral databases, reported references, and verification by commercial standards, 64 glycosides were detected, including 39 glycosides linked with monosaccharides, 18 glycosides linked with disaccharides and 7 glycosides linked with trisaccharides. It is noteworthy that glycosides linked with trisaccharides have previously been rarely reported in tobacco. This method appears to be a useful tool for the systematic screening and characterization of glycosides in tobacco and can potentially be applied to other plants.     

A generic approach to the impurity profiling of drugs using standardised and independent capillary zone electrophoresis methods coupled to electrospray ionisation mass spectrometry

Three standardised, capillary zone electrophoresis-electrospray ionisation mass spectrometry (CZE-ESI-MS) methods were developed for the analysis of six drug candidates and their respective process-related impurities comprising a total of 22 analytes with a range of functional groups and lipophilicities. The selected background electrolyte conditions were found to be: 60/40 v/v 10 mM ammonium formate pH 3.5/organic, 60/40 v/v 10 mM ammonium acetate pH 7.0/organic and 10 mM piperidine, pH 10.5, where the organic solvent is 50/50 v/v methanol/acetonitrile. The coaxial sheath flow consisted of either 0.1% v/v formic acid in 50/50 v/v methanol/water, or 10 mM ammonium acetate in 50/50 v/v methanol/water, depending on the mixture being analysed. Factor analysis and informational theory were used to quantify the orthogonality of the methods and predict their complementarities. The three selected CZE-ESI-MS methods allowed the identification of 21 out of 22 of all the drug candidates and their process-related impurities and provided orthogonality with four established high-performance liquid chromatography-mass spectrometry (HPLC-MS) methods. These methodologies therefore form the basis of a generic approach to impurity profiling of pharmaceutical drug candidates and can be applied with little or no analytical method development, thereby offering significant resource and time savings.     

液相色谱-大气压化学电离离子阱质谱法测定烟草中的游离茄尼醇

用液相色谱/大气压化学电离离子阱质谱建立了一种分析烟草中游离茄尼醇的方法。烟草样品用甲醇振荡提取30 min,在分析前无需进行其它前处理。在1.8μm快速分离C18色谱短柱上用V(甲醇)∶V(异丙醇)=85∶15等梯度洗脱实现了茄尼醇的快速分离。用不带碰撞能量的二级质谱全扫描选择监测离子m/z 613.6进行定量,检出限为0.4μg/L,RSD为1.1%,两种添加量的回收率分别为97%和99%。方法应用于不同烟草和烟草制品样品的检测分析。     

Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling

Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e. 1D SDS-PAGE, 1D preparative SDS-PAGE, IEF-IPG, and 2D-PAGE in their performance as fractionation approaches in nano LC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1D SDS-PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2D-PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1D SDS-PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput.     

Analysis of proteoglycans derived sulphated disaccharides by liquid chromatography/mass spectrometry

A method has been developed for the identification and quantitative determination of sulphated disaccharides derived from chondroitin sulphate (CS) and dermatan sulphate (DS) chains attached to proteoglycans (PGs). After digestion with Chondroitinase ABC, the pool of disaccharides can be directly separated by liquid chromatography on a porous graphitized carbon (PGC) column and identified by on-line electrospray mass spectrometry under negative ionization conditions. The relative intensities of the fragment ions obtained by MS/MS allow to distinguish the sulphate position. Calibration with standard disaccharides allows the quantification of the different isomers. The method showed good repeatability in terms of relative standard deviation (RSD < 2%) and linearity between 0.5 and 50 ng (total injected amount) for both 4- and 6-sulphated disaccharides. The limit of detection achieved in full scan mode was 0.1 ng. The methodology was applied to different types of biological samples obtained from patients suffering from chronic lung inflammation such as: lung tissue, bronchoalveolar lavage fluid (BALF), induced sputum and urine.     

Dansylation isotope labeling liquid chromatography mass spectrometry for parallel profiling of human urinary and fecal submetabolomes

Human urine and feces can be non-invasively collected for metabolomics-based disease biomarker discovery research. Because urinary and fecal metabolomes are thought to be different, analysis of both biospecimens may generate a more comprehensive metabolomic profile that can be better related to the health state of an individual. Herein we describe a method of using differential chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) for parallel metabolomic profiling of urine and feces. Dansylation labeling was used to quantify the amine/phenol submetabolome changes among different samples based on ¹²C-labeling of individual samples and ¹³C-labeling of a pooled urine or pooled feces and subsequent analysis of the ¹³C-/¹²C-labeled mixture by LC-MS. The pooled urine and pooled feces are further differentially labeled, mixed and then analyzed by LC-MS in order to relate the metabolite concentrations of the common metabolites found in both biospecimens. This method offers a means of direct comparison of urinary and fecal submetabolomes. We evaluated the analytical performance and demonstrated the utility of this method in the analysis of urine and feces collected daily from three healthy individuals for 7 days. On average, 2534 ± 113 (n = 126) peak pairs or metabolites could be detected from a urine sample, while 2507 ± 77 (n = 63) peak pairs were detected from a fecal sample. In total, 5372 unique peak pairs were detected from all the samples combined; 3089 and 3012 pairs were found in urine and feces, respectively. These results reveal that the urine and fecal metabolomes are very different, thereby justifying the consideration of using both biospecimens to increase the probability of finding specific biomarkers of diseases. Furthermore, the CIL LC-MS method described can be used to perform parallel quantitative analysis of urine and feces, resulting in more complete coverage of the human metabolome.     

Metabolic profiling of yeast culture using gas chromatography coupled with orthogonal acceleration accurate mass time-of-flight mass spectrometry: Application to biomarker discovery

Yeast and yeast cultures are frequently used as additives in diets of dairy cows. Beneficial effects from the inclusion of yeast culture in diets for dairy mammals have been reported, and the aim of this study was to develop a comprehensive analytical method for the accurate mass identification of the ‘global’ metabolites in order to differentiate a variety of yeasts at varying growth stages (Diamond V XP, Yea-Sacc and Levucell). Microwave-assisted derivatization for metabolic profiling is demonstrated through the analysis of differing yeast samples developed for cattle feed, which include a wide range of metabolites of interest covering a large range of compound classes. Accurate identification of the components was undertaken using GC-oa-ToFMS (gas chromatography-orthogonal acceleration-time-of-flight mass spectrometry), followed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) for data reduction and biomarker discovery. Semi-quantification (fold changes in relative peak areas) was reported for metabolites identified as possible discriminative biomarkers (p-value <0.05, fold change >2), including d-ribose (four fold decrease), myo-inositol (five fold increase), l-phenylalanine (three fold increase), glucopyranoside (two fold increase), fructose (three fold increase) and threitol (three fold increase) respectively.     

Improving liquid chromatography–tandem mass spectrometry determinations by modifying noise frequency spectrum between two consecutive wavelet-based low-pass filtering procedures

This paper employs one chemometric technique to modify the noise spectrum of liquid chromatography–tandem mass spectrometry (LC–MS/MS) chromatogram between two consecutive wavelet-based low-pass filter procedures to improve the peak signal-to-noise (S/N) ratio enhancement. Although similar techniques of using other sets of low-pass procedures such as matched filters have been published, the procedures developed in this work are able to avoid peak broadening disadvantages inherent in matched filters. In addition, unlike Fourier transform-based low-pass filters, wavelet-based filters efficiently reject noises in the chromatograms directly in the time domain without distorting the original signals. In this work, the low-pass filtering procedures sequentially convolve the original chromatograms against each set of low pass filters to result in approximation coefficients, representing the low-frequency wavelets, of the first five resolution levels. The tedious trials of setting threshold values to properly shrink each wavelet are therefore no longer required. This noise modification technique is to multiply one wavelet-based low-pass filtered LC–MS/MS chromatogram with another artificial chromatogram added with thermal noises prior to the other wavelet-based low-pass filter. Because low-pass filter cannot eliminate frequency components below its cut-off frequency, more efficient peak S/N ratio improvement cannot be accomplished using consecutive low-pass filter procedures to process LC–MS/MS chromatograms. In contrast, when the low-pass filtered LC–MS/MS chromatogram is conditioned with the multiplication alteration prior to the other low-pass filter, much better ratio improvement is achieved. The noise frequency spectrum of low-pass filtered chromatogram, which originally contains frequency components below the filter cut-off frequency, is altered to span a broader range with multiplication operation. When the frequency range of this modified noise spectrum shifts toward the high frequency regimes, the other low-pass filter is able to provide better filtering efficiency to obtain higher peak S/N ratios. Real LC–MS/MS chromatograms, of which typically less than 6-fold peak S/N ratio improvement achieved with two consecutive wavelet-based low-pass filters remains the same S/N ratio improvement using one-step wavelet-based low-pass filter, are improved to accomplish much better ratio enhancement 25-folds to 40-folds typically when the noise frequency spectrum is modified between two low-pass filters. The linear standard curves using the filtered LC–MS/MS signals are validated. The filtered LC–MS/MS signals are also reproducible. The more accurate determinations of very low concentration samples (S/N ratio about 7–9) are obtained using the filtered signals than the determinations using the original signals.     

Development of chemical isotope labeling liquid chromatography mass spectrometry for silkworm hemolymph metabolomics

Silkworm (Bombyx mori) is a very useful target insect for evaluation of endocrine disruptor chemicals (EDCs) due to mature breeding techniques, complete endocrine system and broad basic knowledge on developmental biology. Comparative metabolomics of silkworms with and without EDC exposure offers another dimension of studying EDCs. In this work, we report a workflow on metabolomic profiling of silkworm hemolymph based on high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) and demonstrate its application in studying the metabolic changes associated with the pesticide dichlorodiphenyltrichloroethane (DDT) exposure in silkworm. Hemolymph samples were taken from mature silkworms after growing on diet that contained DDT at four different concentrations (1, 0.1, 0.01, 0.001 ppm) as well as on diet without DDT as controls. They were subjected to differential ¹²C-/¹³C-dansyl labeling of the amine/phenol submetabolome, LC-UV quantification of the total amount of labeled metabolites for sample normalization, and LC-MS detection and relative quantification of individual metabolites in comparative samples. The total concentration of labeled metabolites did not show any significant change between four DDT-treatment groups and one control group. Multivariate statistical analysis of the metabolome data set showed that there was a distinct metabolomic separation between the five groups. Out of the 2044 detected peak pairs, 338 and 1471 metabolites have been putatively identified against the HMDB database and the EML library, respectively. 65 metabolites were identified by the dansyl library searching based on the accurate mass and retention time. Among the 65 identified metabolites, 33 positive metabolites had changes of greater than 1.20-fold or less than 0.83-fold in one or more groups with p-value of smaller than 0.05. Several useful biomarkers including serine, methionine, tryptophan, asymmetric dimethylarginine, N-Methyl-D-aspartic and tyrosine were identified. The changes of these biomarkers were likely due to the disruption of the endocrine system of silkworm by DDT. This work illustrates that the method of CIL LC-MS is useful to generate quantitative submetabolome profiles from a small volume of silkworm hemolymph with much higher coverage than conventional LC-MS methods, thereby facilitating the discovery of potential metabolite biomarkers related to EDC or other chemical exposure.     

Liquid chromatography with tandem mass spectrometry quantification of urinary proanthocyanin A2 dimer and its potential use as a biomarker of cranberry intake

The lack of a biomarker for the consumption of cranberries has confounded the interpretation of several studies investigating the effect of cranberry products, especially juices, on health outcomes. The objectives of this pilot study were to develop a liquid chromatography tandem mass spectrometric method for the quantification of the proanthocyanin dimer A‐2 in human urine and validate urinary proanthocyanin dimer A‐2 as a biomarker of cranberry intake. Five healthy, nonsmoking, premenopausal women (20–30 years of age, body mass index: 18.5–25 kg/m²) were assigned to consume a cranberry beverage containing 140 mg proanthocyanin and 35 kilocalories at 237 mL/day, according to a weekly dosing schedule for 7 weeks. Eleven 24 h and morning spot urine samples each were collected from each subject. A reliable, sensitive method for the detection of proanthocyanin dimer A‐2 in urine using liquid chromatography with tandem mass spectrometry was developed with a limit of quantitation of 0.25 ng/mL and a relative standard deviation of 7.26%, precision of 5.7%, and accuracy of 91.7%. While proanthocyanin dimer A‐2 was quantifiable in urine, it did not appear to be excreted in a concentration that corresponded to the dosing schedule and intake of cranberry juice.     

Metabolomic profiling of human urine in hepatocellular carcinoma patients using gas chromatography/mass spectrometry

With the technique of metabolomics, gas chromatography/mass spectrometry (GC/MS), urine or serum metabolites can be assayed to explore disease biomarkers. In this work, we present a metabolomic method to investigate the urinary metabolic difference between hepatocellular carcinoma (HCC, n = 20) male patients and normal male subjects (n = 20). The urinary endogenous metabolome was assayed using chemical derivatization followed by GC/MS. After GC/MS analysis, 103 metabolites were detected, of which 66 were annotated as known compounds. By a two sample t-test statistics with p < 0.05, 18 metabolites were shown to be significantly different between the HCC and control groups. A diagnostic model was constructed with a combination of 18 marker metabolites or together with alphafetoprotein, using principal component analysis and receiver-operator characteristic curves. The multivariate statistics of the diagnostic model yielded a separation between the two groups with an area under the curve value of 0.9275. This non-invasive technique of identifying HCC biomarkers from urine may have clinical utility.     

Alterations of the exo- and endometabolite profiles in breast cancer cell lines: A mass spectrometry-based metabolomics approach

In recent years, knowledge about metabolite changes which are characteristic for the physiologic state of cancer cells has been acquired by liquid chromatography coupled to mass spectrometry. Distinct molecularly characterized breast cancer cell lines provide an unbiased and standardized in vitro tumor model reflecting the heterogeneity of the disease. Tandem mass spectrometry is a widely applied analytical platform and highly sensitive technique for analysis of complex biological samples. Endo- and exometabolite analysis of the breast cancer cell lines MDA-MB-231, -453 and BT-474 as well as the breast epithelial cell line MCF-10A has been performed using two different analytical platforms: UPLC-ESI-Q-TOF based on a scheduled precursor list has been applied for highlighting of significant differences between cell lines and HPLC-ESI-QqQ using multiple reaction monitoring has been utilized for a targeted approach focusing on RNA metabolism and interconnected pathways, respectively. Statistical analysis enabled a clear discrimination of the breast epithelial from the breast cancer cell lines. As an effect of oxidative stress, a decreased GSH/GSSG ratio has been detected in breast cancer cell lines. The triple negative breast cancer cell line MDA-MB-231 showed an elevation in nicotinamide, 1-ribosyl-nicotinamide and NAD+ reflecting the increased energy demand in triple negative breast cancer, which has a more aggressive clinical course than other forms of breast cancer. Obtained distinct metabolite pattern could be correlated with distinct molecular characteristics of breast cancer cells. Results and methodology of this preliminary in vitro study could be transferred to in vivo studies with breast cancer patients.     

Method development for the determination of selected pesticides on tobacco by high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry

A method was developed for the quantitative determination of alachlor, benalaxyl, clomazone, diflubenzuron, dimethomorph, diphenamid, ethofumesate, metalaxyl, methoprene, metobromuron and piperonyl butoxide on tobacco. The pesticides were extracted with water and methanol from five different types of tobacco. The extracts were purified by partition on an extraction cartridge containing diatomaceous earth. The purified extracts were analysed by reversed-phase high-performance liquid chromatography connected to an atmospheric pressure ionisation–electrospray-triple quadrupole mass spectrometer operating in the positive ion mode. Two different transitions and their relative intensities were monitored for unambiguous identification. All pesticides presented overall recovery rates between 35% and 110%. The trueness is near 100% and the interday precision is below 15%. The limits of quantifications are equal or below the guidance residue levels proposed by the Agrochemical Advisory Committee of CORESTA, an association of organisations having scientific research relative to tobacco.     

Liquid chromatography with tandem mass spectrometry method for the simultaneous determination of multiple sweet mogrosides in the fruits of Siraitia grosvenorii and its marketed sweeteners

A high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry method has been developed and validated for the simultaneous quantification of eight major sweet mogrosides in different batches of the fruits of Siraitia grosvenorii and its marketed sweeteners. The sample preparation procedure and chromatographic and mass spectrographic conditions were optimized. Chromatographic separation was achieved on a reversed‐phase Poroshell 120 SB C₁₈ column in 10.0 min with gradient elution with acetonitrile and water, both of which contained 0.1% formic acid. The multiple reaction monitoring scanning mode was employed for quantification in the negative ion mode. The developed method was validated with acceptable linearity (r² = 0.9984–0.9998) over a wide concentration range, precision (relative standard deviations = 1.09–3.91%), stability (relative standard deviations = 1.21–3.01%), and recovery in the range of 91.22–106.58% (relative standard deviations ≤ 3.79%) under optimum conditions. The proposed method was demonstrated to be simple, rapid, specific, and reliable and was successfully applied for the quality control of the fruits of S. grosvenorii and its marketed sweeteners.     

Fast profiling of anthocyanins in wine by desorption nano-electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) mass spectrometry appears to be a useful technique applicable in different areas (e.g. analysis of pharmaceuticals, identification of biologically active compounds in tissues, imaging mass spectrometry). Its modification termed desorption nano-electrospray (nano-DESI) was tested for analysis of anthocyanins. Acidifying of samples and acidic spray liquid (methanol:water = 75:25 with 0.2% HCOOH) were essential for obtaining good quality spectra. Profiles of main anthocyanins in wine samples, two vintages (2005 and 2007) of three cultivars (Alibernet, Neronet and Rubinet), were successfully acquired. They were in agreement with results of LC/MS experiments (anthocyanins isolated by solid phase extraction were separated by μ-HPLC with gradient elution and detected by ESI-MS). Nano-DESI-MS data also allowed to determine ratio of two cultivars (Neronet and Rubinet) in their mixture and to detect coloring of wine by tenturier or elderberry extract. Detection of main anthocyanins in slices of wine grape, chokeberries and elderberries or in a wine stain on cotton fabric is also presented.     

Impurity Profiling of Dirithromycin by Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry

The aim of this study was to characterize as much as possible the unknown peaks in the chromatogram obtained with a non-volatile LC-UV system, which was published earlier for the separation of dirithromycin and its related substances. For this purpose, each peak eluting from the non-volatile system was collected and transferred to a MS, after performing a desalting process. The desalting procedure uses a XTerra RP C₁₈ column (250 mm x 4.6 mm, 5 µm) and two mobile phases consisting of a mixture of water / 0.1% (v/v) formic acid and a mixture of acetonitrile / 0.1% (v/v) formic acid, respectively. Mass spectral data were acquired on an LCQ ion trap mass spectrometer equipped with an electrospray ionization source (ESI), operating in the positive ion mode. In addition to the thirteen already known compounds, seven new compounds were elucidated. Five impurities showed modifications at the amino group of the desosamine molecule, one an alteration at position C-9 and one a modification at position C-13 of the macrolide ring.