High-performance Liquid Chromatography-quadrupole Time-of-flight Mass Spectrometry Research Articles

Application of a multiclass screening method for veterinary drugs and pesticides using HPLC-QTOF-MS in egg samples

A multi-class screening method for the determination of veterinary drugs and pesticides in eggs was developed by high performance liquid chromatography quadrupole-time-of-flight mass spectrometry (HPLC-QTOF-MS). A single-injection workflow was developed for qualitative analysis, and method validation was conducted in terms of retention time deviation (ΔRt), exact mass error (Δm), selectivity, specificity, precision and trueness. The characteristic fragment ions in information dependent acquisition (IDA) mode were summarized for retrospective and non-target screening. Five of 70 samples were found positive with sulfachloropyridazine, sulfamonomethoxine and fipronil sulfone. Compared with triple-quadrupole (QqQ) MS method, the obtained results showed that QTOF-MS gave comparable results to QqQ-MS with residue concentrations higher than 10 μg·kg−1, whereas QqQ-MS was more powerful at lower concentrations (<1 μg·kg−1). Moreover, the amount of fragment ions acquired by HPLC-QTOF-MS decreased substantially at trace levels, which may be a potential cause of reduced reliability of identification.

The effect of storage temperature on the metabolic profiles derived from chicken eggs

Metabonomic profiling, using High Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (HPLC-Q-ToF-MS), was adopted in this study to uncover differences in the small molecule profiles of egg yolks, between two sets of six eggs that were stored at 5 °C and 23 °C for five weeks. Choline, a compound that has previously been shown to have potential use as a biomarker of egg age, was observed to have a significantly lower abundance in the yolks of eggs that were stored at 5 °C for five weeks compared to eggs stored at 23 °C for the same length of time. A follow-up targeted study observed that the previously discovered trend of increasing choline concentration in egg yolk, with increasing egg age, is not repeatable when the eggs are stored at 5 °C rather than 23 °C. It concluded that, by refrigerating eggs at 5 °C, the accurate prediction of egg age using choline as a biomarker can be prevented.

White poplar: Targeted isolation of pancreatic lipase inhibitors

In order to discover new anti-obesity agents, twenty two natural extracts from medicinal plants and agricultural wastes were screened by pancreatic lipase inhibition assay. The methanolic extract of poplar wood (Populus alba L.), an important raw material for paper industry, showed promising inhibitory activity, its IC50 value was 7.15 μg/mL. Through bio-guided fractionation, the ethyl acetate fraction (E) was found to be the major contributor to the activity, IC50 = 4.7 μg/mL. Hence, it was subjected to bio-guided isolation and nine compounds (1–9) were identified from the active sub-fractions. The isolates were identified by means of one- and two-dimensional nuclear magnetic spectroscopy (1D, 2D-NMR), as well as high-performance liquid chromatography quadrupole time-of flight mass spectrometry (HPLC-QTOF-MS). (+)-Taxifolin (5) and (+)-ampelopsin (4) showed the highest activity, their IC50 values were 23.2 and 46.2 μM, respectively. The results indicated that flavonoids and phenolic acids in poplar wood could serve as potential candidates for development of new anti-obesity agents.

Assessing Chemical Diversity in Psilotum nudum (L.) Beauv., a Pantropical Whisk Fern That Has Lost Many of Its Fern-Like Characters.

Members of the Psilotales (whisk ferns) have a unique anatomy, with conducting tissues but lacking true leaves and roots. Based on recent phyogenies, these features appear to represent a reduction from a more typical modern fern plant rather than the persistence of ancestral features. In this study, extracts of several Psilotum organs and tissues were analyzed by Gas Chromatography – Mass Spectrometry (GC-MS) and High Performance Liquid Chromatography – Quadrupole Time of Flight – Mass Spectrometry (HPLC-QTOF-MS). Some arylpyrones and biflavonoids had previously been reported to occur in Psilotum and these metabolite classes were found to be prominent constituents in the present study. Some of these were enriched and further characterized by Nuclear Magnetic Resonance (NMR) spectroscopy. HPLC-QTOF-MS and NMR data were searched against an updated Spektraris database (expanded by incorporating over 300 new arylpyrone and biflavonoid spectral records) to aid significantly with peak annotation. Principal Component Analysis (PCA) with combined GC-MS and HPLC-QTOF-MS data sets obtained with several Psilotum organs and tissues indicated a clear separation of the sample types. The principal component scores for below-ground rhizome samples corresponded to the vectors for carbohydrate monomers and dimers and small organic acids. Above-ground rhizome samples had principal component scores closer to the direction of vectors for arylpyrone glycosides and sucrose (which had high concentrations in above-and below-ground rhizomes). The unique position of brown synangia in a PCA plot correlated with the vector for biflavonoid glycosides. Principal component scores for green and yellow synangia correlated with the direction of vectors for arylpyrone glycosides and biflavonoid aglycones. Localization studies with cross sections of above-ground rhizomes, using Matrix-Assisted Laser Desorption/Ionization – Mass Spectrometry (MALDI-MS), provided evidence for a preferential accumulation of arylpyrone glycosides and biflavonoid aglycones in cells of the chlorenchyma. Our results indicate a differential localization of metabolites with potentially tissue-specific functions in defenses against biotic and abiotic stresses. The data are also a foundation for follow-up work to better understand chemical diversity in the Psilotales and other members of the fern lineage.

Hematopoietic effect of small molecular fraction of Polygoni multiflori Radix Praeparata in cyclophosphamide-induced anemia mice.

The aim of this study is to investigate the protective effects of a small molecular fraction (SMF) of Polygoni multiflori Radix Praeparata (PMRP) in a cyclophosphamide (CTX) induced anemia mouse model. Small molecular fraction of PMRP was prepared and identified by high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). In pharmacology, we examined the peripheral hemogram and thymus and spleen index. The content of granulocyte-macrophage colony-stimulating factor (GM-CSF) in serum was mensurated by enzyme-linked immunosorbent assay (ELISA); The level of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) in serum and spleen tissue homogenate were detected, and glutathione peroxidase (GSH-PX) was assayed in spleen. The results show that SMF can significantly accelerate the recovery of peripheral hemogram, increase the activity of antioxidant enzymes and GM-CSF in serum and spleen. SMF also increases the number of spleen cells, improves bone marrow pathology. In conclusion, the SMF of PMRP promoted the recovery of hematopoietic function in a CTX-induced anemia mouse, which can support SMF to be used as an adjunct to chemotherapy to counteract its side effects.

Food fingerprinting: Mass spectrometric determination of the cocoa shell content (Theobroma cacao L.) in cocoa products by HPLC-QTOF-MS

The determination of cocoa shell content (Theobroma cacao L.) in cocoa products using a metabolomics approach was accomplished via high performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). The developed method was used to separately analyze the polar and non-polar metabolome of the cocoa testa (cocoa shell) and the cocoa cotyledons (cocoa nibs) of cocoa samples from 15 different geographic origins, harvest years, and varieties in positive and negative ion mode. Potential key metabolites were selected which are exclusively contained in the cocoa shell or with significant higher concentration in the cocoa shell than in the cocoa nibs. The pool of potential key metabolites was filtered by established selection criteria, such as temperature stability, fermentations stability, and independence from the geographic origin. Based on these key metabolites an inverse sparse partial least square regression (SPLS) was used for the prediction of the cocoa shell content.

The use of metabonomics to uncover differences between the small molecule profiles of eggs from cage and barn housing systems

Metabonomic techniques have been used to observe differences in the small molecule profiles of chicken eggs, to work towards the detection, and thus prevention, of fraud regarding the misrepresentation of egg farming systems. High Performance Liquid Chromatography-Quadrupole-Time-of-Flight-Mass Spectrometry (HPLC-Q-ToF-MS) was used to obtain profiles of the small molecules found in the yolks of eggs that were laid by hens in enriched cage systems, and in barn systems. Statistical analysis of these small molecule profiles, including the use of XCMS Online and multivariate statistics, was able to uncover differences between the yolks of cage and barn eggs. Several unidentified compounds were found to be present in significantly different abundances between cage and barn egg yolks and one of these compounds was tentatively identified, through the use of METLIN, as 1,2-dipalmitoyl-glycero-3-phosphocholine.

Detection of New Quorum Sensing N-Acyl Homoserine Lactones From Aeromonas veronii.

Sturgeon is an important fresh water-culture fish in China. A problem with sturgeon is its high susceptibility to spoilage. Food spoilage is reported to be regulated by quorum sensing (QS). To identify the QS signals acetylated homoserine lactones (AHLs) in sturgeon and test whether QS plays a role in the spoilage of sturgeon, we investigated the specific spoilage organisms (SSOs) in vacuum packaged sturgeon stored at 4°C and the production of AHLs by sturgeon SSOs. 16S rDNA sequencing and spoilage capabilities analysis revealed that Aeromonas veronii LP-11, Citrobacter freundii LPJ-2, and Raoultella ornithinolytica LPC-3 were the SSOs in sturgeon. Among the three SSOs, only A. veronii LP-11 induced the QS biosensors Agrobacterium tumefaciens KYC55 and Chromobacterium violaceum CV026, suggesting that it produced AHLs. Analysis by thin layer chromatography, high-performance liquid chromatography-triple quadrupole tandem mass spectrometry, and high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC/qTOF-MS) identified that the AHLs produced by A. veronii were C6-SHL, C8-HSL, 3-oxo-C8-HSL, and 3-OH-C8-HSL. Our study revealed that QS system was probably involved in the regulation of sturgeon spoilage and for the first time reported the production of C8-HSL and 3-OH-C8-HSL by genus Aeromonas. As only HPLC/qTOF-MS effectively and accurately identified all the four AHLs produced by A. veronii LP-11, this study also showed that HPLC/qTOF-MS was the most efficient method for rapid analysis of AHLs in complex microbial sample. The study provides new insight into the microbiology of sturgeon spoilage which may be helpful for better sturgeon preservation.

Metabonomic Profiling of Chicken Eggs during Storage Using High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.

Metabonomic techniques have been used to discover subtle differences in the small-molecule profiles of chicken eggs, which could help to combat fraud within the egg industry. High-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-ToF-MS) was used to obtain profiles of the small molecules present in the yolks of chicken eggs stored for different lengths of time. Statistical analysis, including the use of XCMS Online and further exploratory statistics, was able to uncover differences in the abundances of several of the small molecules found in these egg yolks. One of these small molecules was identified through the use of METLIN and MS/MS analysis as choline. A targeted study was then carried out over a longer storage period, using the same instrumentation and analytical techniques, in order to observe how the concentration of choline in egg yolk changes over a longer period of time.

Determination of Branched-Chain Keto Acids in Serum and Muscles Using High Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.

Branched-chain keto acids (BCKAs) are derivatives from the first step in the metabolism of branched-chain amino acids (BCAAs) and can provide important information on animal health and disease. Here, a simple, reliable and effective method was developed for the determination of three BCKAs (α-ketoisocaproate, α-keto-β-methylvalerate and α-ketoisovalerate) in serum and muscle samples using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS). The samples were extracted using methanol and separated on a 1.8 μm Eclipse Plus C18 column within 10 min. The mobile phase was 10 mmol L−1 ammonium acetate aqueous solution and acetonitrile. The results showed that recoveries for the three BCKAs ranged from 78.4% to 114.3% with relative standard deviation (RSD) less than 9.7%. The limit of quantitation (LOQ) were 0.06~0.23 μmol L−1 and 0.09~0.27 nmol g−1 for serum and muscle samples, respectively. The proposed method can be applied to the determination of three BCKAs in animal serum and muscle samples.

The transformation of triclosan by laccase: Effect of humic acid on the reaction kinetics, products and pathway.

This study systematically explored the effect of humic acid (HA) (as model of natural organic matter) on the kinetics, products and transformation pathway of triclosan (TCS) by laccase-catalyzed oxidation. It was found that TCS could be effectively transformed by laccase-catalysis, with the apparent second-order rate constant being 0.056 U-1 mL min-1. HA inhibited the removal rate of TCS. HA-induced inhibition was negatively correlated with HA concentration in the range of 0-10mgL-1 and pH-dependent from 3.5 to 9.5. FT-IR and 13C NMR spectra showed a decrease of aromatic hydroxyl (phenolic) groups and an increase of aromatic ether groups, indicating the cross-linking of HA via C-O-C and C-N-C bonds during enzyme-catalyzed oxidation. Ten principle oxidative products, including two quinone-like products (2-chlorohydroquinone, 2-chloro-5-(2,4-dichlodichlorophenoxy)-(1,4)benzoquinone), one chlorinated phenol (2,4-dichlorophenol (2,4-DCP)), three dimers, two trimmers and two tetramers, were detected by gas chromatograghy/mass spectrometry (GC-MS) and high performance liquid chromatography/quadrupole time-of-flight/mass spectrometry (HPLC/Q-TOF/MS). The presence of HA induced significantly lesser generation of self-polymers and enhanced cross-coupling between HA and self-polymers via C-O-C, C-N-C and C-C coupling pathways. A plausible transformation pathway was proposed as follows: TCS was initially oxidized to form reactive phenoxyl radicals, which self-coupled to each other subsequently by C-C and C-O pathway, yielding self-polymers. In addition, the scission of ether bond was also observed. The presence of HA can promote scission of ether bond and further oxidation of phenoxyl radicals, forming hydroxylated or quinone-like TCS. This study shed light on the behavior of TCS in natural environment and engineered processes, as well provided a perspective for the water/wastewater treatment using enzyme-catalyzed oxidation techniques.

Comprehensive characterization of the in vitro and in vivo metabolites of limonin in human samples using LC-Q-TOF/MS.

Limonin is a bitter triterpenoid dilactone in the genus Citrus with potential medicinal value. In this study, the metabolism of limonin in human was characterized by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). A total of 7 metabolites were identified from human samples. Among them, 3 metabolites of M1, M2 and M4 were detected in urine and feces, and the others were found in intestinal bacteria sample. Notably, M1 and M3 were chemically synthesized, of which the structures were further confirmed by NMR spectra data. The metabolism of limonin involved three major pathways, namely, reduction, hydrolysis and methylation. The reduction and hydrolysis were commonly observed in ring D of limonin. The metabolites showed decomposition in ring A. This study provides data for the metabolism of limonin in humans, and will contribute to explain its biological activity.

Systematic identification of flavonols, flavonol glycosides, triterpene and siraitic acid glycosides from Siraitia grosvenorii using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry combined with a screening strategy

The fruits of Siraitia grosvenorii are considered to be health-promoting because of the diversity of their bioactive ingredients. In the present study, a screening method, using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS) combined with a screening strategy, has been established. The technology was used to systematically screening the targeted metabolites, primarily from the complex matrix of S. grosvenorii. The compounds were then identified by their exact masses and characteristic fragment ions, in comparison with the fragmentation behaviors of 19 references. Finally, 122 compounds, including 53 flavonols and flavonol glycosides, 59 triterpene glycosides and 10 siraitic acid glycosides, were screened and identified in 10-, 50- and 80-day fruits, roots, stems and leaves of S. grosvenorii. 98 of them were reported for the first time. Additionally, the distribution of all identified components in different parts of the plant was determined and metabolic networks for flavonol and triterpene glycosides were proposed.

Highly Selective Bioconversion of Ginsenoside Rb1 to Compound K by the Mycelium of Cordyceps sinensis under Optimized Conditions.

Compound K (CK), a highly active and bioavailable derivative obtained from protopanaxadiol ginsenosides, displays a wide variety of pharmacological properties, especially antitumor activity. However, the inadequacy of natural sources limits its application in the pharmaceutical industry. In this study, we firstly discovered that Cordyceps sinensis was a potent biocatalyst for the biotransformation of ginsenoside Rb1 into CK. After a series of investigations on the biotransformation parameters, an optimal composition of the biotransformation culture was found to be lactose, soybean powder and MgSO4 without controlling the pH. Also, an optimum temperature of 30 °C for the biotransformation process was suggested in a range of 25 °C–50 °C. Then, a biotransformation pathway of Rb1 → Rd → F2 → CK was established using high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). Our results demonstrated that the molar bioconversion rate of Rb1 to CK was more than 82% and the purity of CK produced by C. sinensis under the optimized conditions was more than 91%. In conclusion, the combination of C. sinensis and the optimized conditions is applicable for the industrial preparation of CK for medicinal purposes.

HPLC/Q-TOF-MS-Based Identification of Absorbed Constituents and Their Metabolites in Rat Serum and Urine after Oral Administration of Cistanche deserticola Extract.

As a famous health food in China, Cistanche deserticola (C. deserticola) suggested an estrogenic activity according to our previous study. However, no one clarifies its active material basis to date. To find more potentially active constituents and elucidate metabolic pathways of metabolites, a method to simultaneously analyze multiple absorbed constituents and metabolites from C. deserticola in rat serum and urine was established using high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF-MS). Based on HPLC/Q-TOF-MS method, a total of 24 components, involving 9 prototype constituents and 15 metabolites in rat serum and urine samples, were tentatively identified based on retention time, ultraviolet spectrum, MS data, compound fragmentation laws, published literatures, and reference substances. Most of the compounds existed in the form of metabolites. The proposed metabolic pathways of main metabolites were discussed, including methylation, demethylation, hydrolysis, hydroxylation, acetoxylation, glucuronidation, dehydrogenation, sulfation, esterification, and so on. Phenylethanoid glycosides were extensively metabolized and mutually transformed in vivo. This investigation provided valuable information for further study of the active ingredients and action mechanism of C. deserticola.

Metabolic profile of miltirone in rats by high performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Miltirone is one of the bioactive diterpene quinones isolated from Salvia miltiorrhiza Bunge. This compound has been found to possess significant anticancer, antibacterial, antioxidant, and anti-inflammatory activities. However, the metabolic fate of miltirone remains unknown. In order to explore whether miltirone is extensively metabolized, we investigated the metabolites of miltirone in plasma, bile, urine, and feces samples following oral and intravenous administration to the rats. By using high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF-MS) coupled with mass detect filter (MDF) method, a total of 15 metabolites were identified from the biosamples. Both phase I and phase II metabolites were observed in the metabolic profile and the metabolic pathways involved in reduction, oxidation, monohydroxylation, dihydroxylation, glucuronidation and sulfation. The results indicated that hepatocyte metabolism was the major route of clearance for the parent compound. The present study provided valuable information for better understanding of the efficacy and safety of miltirone.

An improved separation method for classification of Macleaya cordata from different geographical origins

In this study, an improved separation method for the metabolomic study of polar and basic secondary metabolites by high-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (HPLC-Q-TOF-MS) using an “Xcharge C18” column has been developed.

HPLC-QTOF-MS Method for Identification and Determination of Bleomycin A2 and B2 Fractions

Bleomycin is a very important drug in oncology used as first-line treatment for many cancers. The development of high-performance liquid chromatography—tandem mass spectrometry method for the separation, identification, and determination of two major structurally related analogs of this antibiotic drug, namely bleomycin A2 and B2, is presented in this work. The proposed method is based on a hydrophilic interaction chromatography (HILIC) stationary phase that enables one to avoid an ion-pairing reagent in the separation system (formerly used in mobile phase for bleomycin) in order to properly separate these highly polar bleomycin analogs. The ion-pairing reagent-free HILIC separation system is suitable for coupling the chromatographic and mass spectrometry stages (for the first time for bleomycin). Some performance parameters, namely, limit of detection (ng/mL-pg/mL), limit of quantification, linearity, precision, and recovery/accuracy, were evaluated showing high reliability, selectivity, and sensitivity of the method. It was successfully applied for the quality drug control determining bleomycin A2 and B2 fractions in commercial pharmaceuticals (Bleomedac infusions). In addition, the high-performance liquid chromatography–quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was able to determine an accurate molecular weight of bleomycin A2 and B2 fractions, confirming an identity/quality of the (commercial) drug. The possibilities of the HPLC-QTOF-MS method to identify bleomycin A2 and B2 in model plasma samples were also demonstrated.

Liquid chromatography-tandem mass spectrometry-based research on serum metabonomics of lung cancer patients

Objective To explore the differences in serum metabolism between patients with lung cancer and healthy people using the technology of metabolomics.Methods Serum samples from 30 lung cancer patients and 30 healthy people were analyzed by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS).Orthogonal to partial least squares discriminant analysis was performed for modeling.Two sample t-test was used to identify differences in metabolite concentrations.Results Multivariate statistical analysis yielded a significant distinction in the metabolic profiles between lung cancer patients and healthy people.The t-test results indicated a total of 36 metabolites in the serum with statistically significant differences.Conclusion The method of lung cancer serum metabolomics study has been constructed using the technology of HPLC-Q-TOF/MS. Key words: Lung cancer; High performance liquid chromatography-quadrupole time-of-flight mass spectrometry; Metabonomics

Secondary organic aerosol formation and composition from the photo-oxidation of methyl chavicol (estragole)

Abstract. The increasing demand for palm oil for uses in biofuel and food products is leading to rapid expansion of oil palm agriculture. Methyl chavicol (also known as estragole and 1-allyl-4-methoxybenzene) is an oxygenated biogenic volatile organic compound (VOC) that was recently identified as the main floral emission from an oil palm plantation in Malaysian Borneo. The emissions of methyl chavicol observed may impact regional atmospheric chemistry, but little is known of its ability to form secondary organic aerosol (SOA). The photo-oxidation of methyl chavicol was investigated at the European Photoreactor chamber as a part of the atmospheric chemistry of methyl chavicol (ATMECH) project. Aerosol samples were collected using a particle into liquid sampler (PILS) and analysed offline using an extensive range of instruments including; high-performance liquid chromatography mass spectrometry (HPLC-ITMS), high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-QTOFMS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The SOA yield was determined as 18 and 29% for an initial VOC mixing ratio of 212 and 460 ppbv (parts per billion by volume) respectively; using a VOC:NOx ratio of ~5:1. In total, 59 SOA compounds were observed and the structures of 10 compounds have been identified using high-resolution tandem mass spectrometry. The addition of hydroxyl and/or nitro-functional groups to the aromatic ring appears to be an important mechanistic pathway for aerosol formation. This results in the formation of compounds with both low volatility and high O:C ratios, where functionalisation rather than fragmentation is mainly observed as a result of the stability of the ring. The SOA species observed can be characterised as semi-volatile to low-volatility oxygenated organic aerosol (SVOOA and LVOOA) components and therefore may be important in aerosol formation and growth.