Prediction of Japanese green tea ranking by gas chromatography/mass spectrometry-based hydrophilic metabolite fingerprinting

An innovative technique for green tea's quality determination was developed by means of metabolomics. Gas-chromatography coupled with time-of-flight mass spectrometry and multivariate data analysis was employed to evaluate the quality of green tea. Alteration of green tea varieties and manufacturing processes effects a variation in green tea metabolites, which leads to a classification of the green tea's grade. Therefore, metabolic fingerprinting of green tea samples of different qualities was studied. A set of ranked green tea samples from a Japanese commercial tea contest was analyzed with the aim of creating a reliable quality-prediction model. Several multivariate algorithms were performed. Among those, the partial least-squares projections to latent structures (PLS) analysis with the spectral filtering technique, orthogonal signal correction (OCS), was found to be the most practical approach. In addition, metabolites that play an important role in green tea's grade classification were identified.     

Quality prediction of Japanese green tea using pyrolyzer coupled GC/MS based metabolic fingerprinting

A couple between pyrolyzer and gas chromatography/mass spectrometry (GC/MS) has allowed a fast, simple, and low-cost approach to evaluate a quality of Japanese green tea without any sample preparation or derivatization techniques. Using our method, errors from sample preparation could be avoided since raw samples were directly extracted through the extreme heat of the pyrolyzer. In addition, undesired reactions from expensive derivatizing agents, which are commonly needed to treat the samples before analyzing with GC/MS, could be omitted. In order to illustrate the efficiency of this technique, a set of green tea samples from the Tea contest in 2005 in the Kansai area were used. Projection to latent structure by means of partial least squares (PLS) along with orthogonal signal correction (OSC) was selected to explain the relation between green tea's metabolite profiling and its quality. The quality of the model was validated by testing and comparing the predictive ability to the respective model.     

Confirmation of the allergenic peanut protein, Ara h 1, in a model food matrix using liquid chromatography/tandem mass spectrometry (LC/MS/MS)

Enzymatic digestion of total protein along with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to confirm the presence of a major peanut allergen in food. Several peptides obtained from the enzymatic digestion of the most abundant peanut allergen, Ara h 1, were identified as specific peptide biomarkers for peanut protein. Using ice cream as a model food matrix, a method was developed for the detection of the allergen peptide biomarkers. A key component of the method was the use of molecular mass cutoff filters to enrich the Ara h 1 in the protein extracts. By applying the method to ice cream samples containing various levels of peanut protein, levels as low as 10 mg/kg of Ara h 1 could routinely be detected. This method provides an unambiguous means of confirming the presence of the peanut allergen, Ara h 1, in foods and can easily be modified to detect other food allergens.     

Simple and sensitive method for pyrroloquinoline quinone (PQQ) analysis in various foods using liquid chromatography/electrospray-ionization tandem mass spectrometry

Pyrroloquinoline quinone (PQQ) is believed to be an important factor for mammalian growth and development and has, therefore, been declared a vitamin by some researchers. However, this issue remains controversial, and from a nutritional viewpoint, accurate determination of PQQ levels in a variety of foods is very important. Here, we describe a simple, highly sensitive, and highly selective method for quantitative analysis of PQQ. Liquid foods or aqueous extracts of solid foods were analyzed using high-performance liquid chromatography (HPLC) combined with electrospray-ionization (ESI) tandem mass spectrometry (MS/MS). (15)N-labeled PQQ was added to the samples as an internal standard. Quantitative analyses of PQQ were performed by multiple reaction monitoring (MRM) with LC/MS/MS. Free PQQ was detected in almost all food samples in the range 0.19-7.02 ng per g fresh weight (for solid foods) or per mL (liquid foods). This method will enable the rapid and simple determination of PQQ levels in many samples.     

Development of a liquid chromatography-tandem mass spectrometry method using capillary liquid chromatography and nanoelectrospray ionization-quadrupole time-of-flight hybrid mass spectrometer for the detection of milk allergens

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the tryptic digest of a cleaned-up food matrix extract was used for the detection of milk allergens. The emphasis of this study was on casein, which is the most abundant milk protein and is also considered the most allergenic. A sample cleanup method was developed using an ion exchange column and centriprep device. Cookies spiked with milk powder from 0 to 1250 ppm were extracted, cleaned up, and either digested directly by trypsin or further cleaned up by gel electrophoresis before digestion. The peptide mixture was analyzed on a capillary LC-quadrupole time-of-flight system. Two marker peptides from alphaS1-casein were identified and used for prescreening. The MS/MS data from the mass spectrometry system were processed with Masslynx v4.0 and submitted for database search using either ProteinLynx Global Server or Mascot for protein identification. The LC-MS/MS method, using casein enzyme-linked immunosorbent assay as a reference, was tested on the cookie matrix and was extended to other sample matrices. There were good agreements between the two. This LC-MS/MS method provides a valuable confirmatory method for the presence of casein. It also allows the simultaneous detection of other milk allergens.     

Confirmation of peanut protein using peptide markers in dark chocolate using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Detection of peptides from the peanut allergen Ara h 1 by liquid chromatography-mass spectrometry (LC-MS) was used to identify and estimate total peanut protein levels in dark chocolate. A comparison of enzymatic digestion subsequent to and following extraction of Ara h 1 from the food matrix revealed better limits of detection (LOD) for the pre-extraction digestion (20 ppm) than for the postextraction digestion (50 ppm). Evaluation of LC-MS instruments and scan modes showed the LOD could be further reduced to 10 ppm via a triple-quadrupole and multiple-reaction monitoring. Improvements in extraction techniques combined with an increase in the amount of chocolate extracted (1 g) improved the LOD to 2 ppm of peanut protein. This method provides an unambiguous means of confirming the presence of the peanut protein in foods using peptide markers from a major allergen, Ara h 1, and can easily be modified to detect other food allergens.     

Identification of procyanidins in cocoa (Theobroma cacao) and chocolate using high-performance liquid chromatography/mass spectrometry

Monomeric and oligomeric procyanidins present in cocoa and chocolate were separated and identified using a modified normal-phase high-performance liquid chromatography (HPLC) method coupled with on-line mass spectrometry (MS) analysis using an atmospheric pressure ionization electrospray chamber. The chromatographic separation was achieved using a silica stationary phase in combination with a gradient ascending in polarity. This qualitative report confirms the presence of a complex series of procyanidins in raw cocoa and certain chocolates using HPLC/MS techniques. Although both cocoa and chocolate contained monomeric and oligomeric procyanidin units 2-10, only use of negative mode provided MS data for the higher oligomers (i.e., >pentamer). Application of this method for qualitative analysis of proanthocyanidins in other food products and confirmation of this method as a reliable quantitative tool for determining levels of procyanidins in cocoa, chocolate, and other food products are currently being investigated.     

Characterization of grape procyanidins using high-performance liquid Chromatography/Mass spectrometry and matrix-assisted laser Desorption/Ionization time-of-flight mass spectrometry

High-performance liquid chromatography/mass spectrometry (HPLC/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to characterize the procyanidin composition of the grape seed extract. The detection of the oligomers composed of (+)-catechin, (-)-epicatechin, and their galloylated derivatives in the grape seeds is demonstrated. With MALDI-TOF MS, oligomers up to nonamers were observed. The potential of the MALDI-TOF MS technique as a quantification tool is also discussed. The information presented in this study could lead to the determination of procyanidin content and their molecular weight distribution in grape seeds.     

Identification of olive (Olea europaea) pulp proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-liquid chromatography tandem mass spectrometry

Proteins in the pulp of olive ( Olea europaea ) constitute a minor fraction. They have been sparsely studied despite their suggested role in oil stability and olive allergenicity. The analysis of a pulp protein extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a major band at 24 kDa that was subjected to tryptic in-gel digestion. Peptide extracts were analyzed by MALDI-TOF MS and nanoLC-MS/MS. The use of different search engines enabled the assignment of a number of fragmentation spectra to peptide sequences, identifying a major band as a thaumatin-like protein and other low-abundant proteins such a drought-induced protein SDi-6-like, an acyl carrier protein, Cu/Zn and Mn superoxide dismutases, a small heat shock protein, and an ATP-dependent protease subunit. Many of the produced spectra did not give good matches in the database searches, due to the scarce presence of O. europaea entries in protein databases. Nevertheless, a huge number of spectra corresponded to peptides, which showed a high degree of homology with others from sequenced organisms. These results proved that database searching with MS/MS spectra constitutes a promising approach for the characterization of olive pulp proteins.     

Analysis of veterinary drug residues in frog legs and other aquacultured species using liquid chromatography quadrupole time-of-flight mass spectrometry

A liquid chromatography quadrupole time-of-flight (Q-TOF) mass spectrometry method was developed to analyze veterinary drug residues in frog legs and other aquacultured species. Samples were extracted using a procedure based on a method developed for the analysis of fluoroquinolones (FQs) in fish. Briefly, the tissue was extracted with dilute acetic acid and acetonitrile with added sodium chloride. After centrifugation, the extracts were evaporated and reconstituted in mobile phase. A molecular weight cutoff filter was used to clean up the final extract. A set of target compounds, including trimethoprim, sulfamethoxazole, chloramphenicol, quinolones, and FQs, was used to validate the method. Screening of residues was accomplished by collecting TOF (MS1) data and comparing the accurate mass and retention times of compounds to a database containing information for veterinary drugs. An evaluation of the MS data in fortified frog legs indicated that the target compounds could be consistently detected at the level of concern. The linearity and recoveries from matrix were evaluated for these analytes to estimate the amount of residue present. MS/MS data were also generated from precursor ions, and the mass accuracy of the product ions for each compound was compared to theoretical values. When the method was used to analyze imported frog legs, many of these residues were found in the samples, often in combination and at relatively high concentrations (>10 ng/g). The data from these samples were also evaluated for nontarget analytes such as residue metabolites and other chemotherapeutics.     

Quantification of ptaquiloside and pterosin B in soil and groundwater using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The carcinogenic compound ptaquiloside is produced by bracken fern (Pteridium aquilinum L.). Ptaquiloside can enter the soil matrix and potentially leach to the aquatic environment, and methods for characterizing ptaquiloside content and fate in soil and groundwater are needed. A sensitive detection method has been developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analyzing ptaquiloside and its transformation product pterosin B. Detection limits are 0.19 microg/L (ptaquiloside) and 0.15 microg/L (pterosin B), which are 300-650 times better than previously published LC-UV methods. Sequential soil extractions are made using 5 mM ammonium acetate for extraction of ptaquiloside, followed by 80% methanol extraction for pterosin B. Groundwater samples are cleaned-up and preconcentrated by a factor of 20 using solid-phase extraction. The LC-MS/MS method enables quantification of ptaquiloside and pterosin B in soil and groundwater samples at environmentally relevant concentrations and delivers a reliable identification because of the structure-specific detection method.     

稳定性肥料中硝化抑制剂作用效果的检测方法

以硝化抑制率作为评价指标,研究了影响硝化抑制剂抑制效果测定方法的主要因素,包括氮土比、培养时间以及土壤类型等。确立了测定稳定性肥料中硝化抑制剂抑制作用效果的最佳检测方法:称取风干后的棕壤200 g,以氮土比1.15∶1 000准确称取样肥,并将其充分混匀,以25%的含水量,在30℃培养箱中培养。选择自培养开始的第9、12、15 d测定土壤中硝态氮(包括亚硝态氮)的质量分数。该检测方法提高了评价效率和准确度。     

Multiresidue analysis of seven anticoagulant rodenticides by high-performance liquid chromatography/electrospray/mass spectrometry

Mice and rat populations are commonly controlled by two classes of rodenticide anticoagulants, coumarins and indandiones. However, poisoning of nontarget animals also often occurs. For cases such as these, a rapid, multiresidue method, which provides positive confirmation for both classes of anticoagulant rodenticides, is needed by diagnostic laboratories. A method was developed for the determination of seven anticoagulant rodenticides, coumafuryl, pindone, warfarin, diphacinone, chlorophacinone, bromadiolone, and brodifacoum, in diverse matrices, animal feed, cooked beef, and fruit-flavored beverages using high-performance liquid chromatography/electrospray/mass spectrometry. Detection was by MS/MS with electrospray ionization in negative mode. Confirmation was by retention time, m/z of molecular ion, and two parent-daughter transitions. Recoveries from selected the matrices ranged from 61 to 117%. Limits of quantitation were as low as 1.5-4.5 ng g-1. The developed method was rapid and provided the simultaneous confirmation and quantification of the seven anticoagulant rodenticides.     

Metabolite profiling using (1)H NMR spectroscopy for quality assessment of green tea, Camellia sinensis (L.)

A set of 191 green teas from different countries was collected and analyzed by (1)H NMR. It was proposed to establish if the teas could be discriminated according to the country of origin or with respect to quality. Both principal component analysis (PCA) and cluster analysis were applied to the data. Some separation of Chinese and non-Chinese teas was observed. The present results did not allow allocation of samples to individual countries, but cluster analysis suggested that it might be possible with an augmented sample set. The PCA did show a separation between the Longjing type (highest quality Chinese tea) and most other Chinese teas and indicated some metabolites that could be responsible for the difference. Longjing teas showed higher levels of theanine, gallic acid, caffeine, epigallocatechin gallate, and epicatechin gallate and lower levels of epigallocatechin when compared with other teas. These compounds have been mentioned previously in connection with quality, but it was also shown that higher levels of theogallin (5-galloyl quinic acid), theobromine, 2-O-(beta-l-arabinopyranosyl)-myo-inositol and some minor sugar-containing compounds were found in Longjing teas while higher levels of fatty acids and sucrose were found in the other teas. These new markers could prove to be useful for the authentication of bulk tea.     

Monitoring of artemisinin, dihydroartemisinin, and artemether in environmental matrices using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The area cultivated with Artemisia annua for the extraction of the antimalarial compound artemisinin is increasing, but the environmental impact of this cultivation has not yet been studied. A sensitive and robust method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of artemisinin in soil. Dihydroartemisinin and artemether were included in the method, and performance on analytical columns of both traditional C(18) phenyl-hexyl and porous shell particles-based Kinetex types was characterized. The versatility of the method was demonstrated on surface water and groundwater samples and plant extracts. The limit of detection was 55, 30 (25 ng/g soil), and 4 ng/mL for dihydroartemisinin, artemisinin, and artemether, respectively. Method performance was demonstrated using naturally contaminated soil samples from A. annua fields in Kenya. The highest observed concentrations were above EC(10) for lettuce growth. Monitoring of artemisinin in soil with A. annua crop production seems necessary to further understand the impact in the environment.     

Identification of flavonoids in Hakmeitau beans (Vigna sinensis) by high-performance liquid chromatography-electrospray mass spectrometry (LC-ESI/MS)

Liquid chromatography coupled with electrospray mass spectrometry (LC-ESI/MS) with positive and negative ion detection was used for the identification of flavonoids in Hakmeitau beans, a black seed cultivar of cowpea (Vigna sinensis). Gradient elution with water and acetonitrile, both containing 2% formic acid, was employed in chromatographic separation. The peaks were identified by comparison of the retention times and the UV-vis spectroscopic and mass spectrometric data with authentic standards and/or literature data. The identified flavonoids included six anthocyanins (cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, malvidin 3-O-glucoside, peonidin 3-O-glucoside, and petunidin 3-O-glucoside) and four flavonol/flavonol glycosides (kaempferol 3-O-glucoside, quercetin, quercetin 3-O-glucoside, and quercetin 3-O-6' '-acetylglucoside). The tentatively identified flavonoids included two anthocyanins (malvidin 3-O-acetylglucoside and peonidin 3-O-malonylglucoside) and three flavonol glycosides (myricetin-3-O-glucoside, quercetin 7-O-glucoside, and quercetin-3-O-diglucoside). These flavonoids are present in seed coats, and the contents of anthocyanins and flavonol glycosides were 20.7 and 2.0 mg/g, respectively.     

Determination of 2-alkylcyclobutanones with electronic impact and chemical ionization gas chromatography/mass spectrometry (GC/MS) in irradiated foods

The use of a column containing 60 g of silica gel for cleanup and the use of isobutane as a reactant reagent for chemical ionization-mass spectrometric analysis of the saturated and monounsaturated alkyl side-chain 2-alkylcyclobutanones (2-ACBs; specifically induced by irradiation from fat in foods until the proof of contrary) has improved both the sensibility and the selectivity of the method when applied for the detection of irradiated foods. The quality of the chromatograms obtained was improved, allowing the detection of food samples (avocados) irradiated at low doses (0.1 kGy) or irradiated ingredients included in low proportions (less than 5%, wt/wt) in nonirradiated culinary foods. These analytical modifications for the detection of 2-ACBs on the official EN 1785 method enable an extension of its current field of application using common equipment of food quality control laboratories.     

Determination of the 1'S and 1'R diastereomers of metolachlor and S-metolachlor in water by chiral liquid chromatography-mass spectrometry/mass spectrometry (LC/MS/MS)

An enantioselective method for the separation and quantification of the diastereomer pairs of metolachlor and S-metolachlor in surface and ground waters is presented. Samples are purified and concentrated using a C18 (octadecyl silica) solid-phase extraction (SPE) procedure and analyzed by chiral column liquid chromatography-mass spectrometry/mass spectrometry (LC/MS/MS) interfaced with either atmospheric pressure chemical ionization (APcI) or atmospheric pressure photoionization (APPI) sources. The overall mean percent procedural recoveries (percent relative standard deviations) are 89% (10.6%) for surface water and 80% (9.1%) for ground water. The method limit of quantitation (LOQ) is 0.10 ppb. The method validation was conducted under U.S. EPA FIFRA Good Laboratory Practice Guidelines 40 CFR 160.     

Determination of flunixin in edible bovine tissues using liquid chromatography coupled with tandem mass spectrometry

An accurate, reliable, and reproducible assay was developed and validated to determine flunixin in bovine liver, kidney, muscle, and fat. The overall recovery and percent coefficient of variation (%CV) of twenty-eight determinations in each tissue for flunixin free acid were 85.9% (5.9% CV) for liver, 94.6% (9.9% CV) for kidney, 87.4% (4.7% CV) for muscle, and 87.6% (4.4% CV) for fat. The theoretical limit of detection was 0.1 microg/kg (ppb, ng/g) for liver and kidney, and 0.2 ppb for muscle and fat. The theoretical limit of quantitation was 0.3, 0.2, 0.6, and 0.4 ppb for liver, kidney, muscle, and fat, respectively. The validated lower limit of quantitation was 1 ppb for edible tissues with the upper limit of 400 ppb for liver and kidney, 100 ppb for fat, and 40 ppb for muscle. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. Briefly, the method involves an initial acid hydrolysis, followed by pH adjustment ( approximately 9.5) and partitioning with ethyl acetate. A portion of the ethyl acetate extract was purified by solid-phase extraction using a strong cation exchange cartridge. The eluate was then evaporated to dryness, reconstituted, and analyzed using LC/MS/MS. The validated method is sensitive and specific for flunixin in edible bovine tissue.     

Development of liquid chromatography mass spectrometry method for analysis of organic N monomers in soil

The aim of this study was to develop an analytical procedure based on liquid chromatography-mass spectrometry (LC–MS) for analysis of monomeric organic N compounds in soil extracts. To benchmark the developed LC–MS method it was compared with a capillary electrophoresis–mass spectrometry (CE–MS) method recently used for analysis of small organic N monomers in soil. The separation was optimized and analytical performance assessed with 69 purified standards, then the LC–MS method was used to analyse soil extracts. Sixty-two out of 69 standards were analysable by LC–MS with separation on a hydrophilic interaction liquid chromatography column. The seven compounds that could not be analysed were strongly cationic polyamines. Limits of detection for a 5 μL injection ranged between 0.002 and 0.38 μmol L⁻¹, with the majority (49 out of 62) having limits of detection better than 0.05 μmol L⁻¹. The overall profile and concentration of small organic N monomers in soil extracts was broadly similar between LC–MS and CE–MS, with the notable exception of four ureides that were detected by LC–MS only. In soil extracts that had been concentrated ten-fold the detection and quantification of (some) organic N compounds was compromised by the presence of large amounts of inorganic salts. The developed LC–MS method offered advantages and disadvantages relative to CE–MS, and a combination of the two methods would achieve the broadest possible coverage of organic N in soil extracts.