LC QTOF Research Articles

Transformation profiles of the isoflavones in germinated soybean based on UPLC–DAD quantification and LC–QTOF–MS/MS confirmation

Germinated soybean is one kind of food and a medicine. In the actual process of producing a large amount of naturally germinated soybean, it is difficult to strictly control the germination process conditions. However, sprout length may be more suitable as the terminal judgment indicator for naturally germinated soybean. An UPLC–DAD method was developed and validated to explore the transformation profiles of soybean isoflavones in germinated yellow or black soybean with different sprout lengths. Moreover, an LC − QTOF−MS/MS method was used to avoid false positive results. The contents of daidzein, glycitein, and genistein almost reached their corresponding maximum values when the sprout length ranged from 1.0 cm to 1.5 cm (P < 0.05). Therefore, yellow soybean is suggested to be the processing raw material with higher contents of those isoflavones, and the optimal sprout length for germinated soybean may be in the range of 1.0–1.5 cm.

Insight into the relationship between metabolic enzymes and oxadiazon degradation in Oryza sativa for reducing environmental risks

Oxadiazon (ODZ) is extensively utilized in agricultural fields for weed control owing to its strong effectiveness. However, excessive loading of ODZ in water bodies and agricultural soils can lead to various environmental concerns. Therefore, it is crucial to understand the ODZ metabolic process and associated mechanisms in crops to assess the likelihood of ODZ contamination in the environment. This study aimed to assess the effects of ODZ on the growth and toxicological responses of rice (Oryza sativa). The growth of rice tissues was notably compromised with the increase in ODZ concentrations. RNA sequencing in combination with liquid chromatography–quadrupole-time-of-flight–high-resolution mass spectrometry/mass spectrometry (LC–Q-TOF–HRMS/MS) analysis allowed for the identification of numerous transcriptional components associated with ODZ metabolism. Four libraries comprising rice roots and shoots exposed to ODZ were RNA-sequenced in triplicate. The application of environmentally realistic ODZ concentrations upregulated the expression of 844 genes in shoots and 1476 genes in roots. Gene enrichment analysis revealed the presence of multiple enzymes involved in ODZ metabolism and detoxification. These enzymes play a critical role in mitigating environmental stress and facilitating xenobiotic metabolism. Notably, among differentially expressed genes, several key enzymes were identified, including cytochrome P450s, protein kinases, aminotransferases, and ATP-binding cassette transporters involved in the metabolic process. Using LC–Q-TOF–HRMS/MS, 3 metabolites and 13 conjugates were identified in multiple metabolic pathways involving oxidation, hydrolysis, glycosylation, acetylation, and methylation. This study successfully established a potential link between the specific metabolic products of ODZ and increased activities of their corresponding enzymes. Moreover, this study considerably elucidates the detailed pathways and mechanisms involved in ODZ metabolism. The study findings provide valuable insights into the development of genotypes for reducing ODZ residues in paddy fields and minimizing their accumulation in rice crops.

A novel liquid chromatography with quadrupole time-of-flight-tandem mass spectroscopy method for ultra-trace level identification and quantification of the genotoxic impurity 2,6-diamino-5-nitropyrimidin-4(3H)-one in valganciclovir hydrochloride.

In the present study, the main objective is to develop an analytical method for ultra-trace level measurement of 2,6-diamino-5-nitropyrimidin-4(3H)-one (DMNP) in valganciclovir hydrochloride (VAL) using liquid chromatography-quadrupole time-of-flight-tandem mass spectroscopy (LC-QTOF-MS/MS). In the early stages of guanine synthesis, DMNP is formed, and guanine is known to be the key starting material for the synthesis of VAL. Taking into consideration DMNP potential genotoxicity, this analytical method has been developed. This method is time saving and suitable for confirming the masses of parent and fragment ions by MS and MS/MS further fragmentation. An isocratic program and Acquity UPLC HSS cyano column (100 ×2.1mm ×1.8μm) were used to achieve optimal separation between VAL and the DMNP impurity. A 0.1% ammonia solution in Milli-Q water was used as mobile phase A, and methanol was used as mobile phase B in the ratio 90:10v/v in isocratic mode. In accordance with the International Conference on Harmonization's requirements, the developed method was validated. The detection and quantification levels were found to be 0.028 and 0.083 ppm respectively. The DMNP impurity is linear from 0.083 to 1.245 ppm levels with correlation coefficient (R2 ) of 0.9960. The recoveries were found to be 97.0-107.9%.

Exploring the Oxidative Effects of the Microbial Electro-Fenton Process on the Depolymerization of Lignin Extracted from Rice Straw in a Bio-Electrochemical System Coupled with Wastewater Treatment.

Lignin is a potential renewable feedstock to produce value-added compounds, but the overwhelming bulk of it is either burned for energy or discarded as waste. This paper addressed two critical issues: waste-to-value generation and management by demonstrating the in situ depolymerization of lignin extracted from waste rice straw utilizing the microbial electro-Fenton process in a microbial peroxide-producing cell (MPPC), a type of bio-electrochemical cell, for value addition while synchronously treating wastewater. The MPPC electrochemical voltage yields of 0.171 ± 0.05-0.497 ± 0.2 V produced 9 ± 0.43-34 ± 0.11 mM of H2O2, which was utilized to depolymerize lignin at various concentrations. Interestingly, a direct correlation was observed between lignin depolymerization and H2O2 concentration, while Fourier-transform infrared spectroscopy data revealed a constant disruption of the lignin structure accurately in the wavenumber region of 1000-1750 cm-1 irrespective of the H2O2 concentration. Carboxylic acid derivatives, benzopyran, hexanoic acid, and other valuable compounds were detected in the LC QTOF MS data from the depolymerized lignin mixture. Remarkably, SEM analysis demonstrated morphological changes in depolymerized lignin induced by the oxidative effects of hydroxyl radicals. Biochemical oxygen demand and chemical oxygen demand removal was 60 ± 3-85 ± 1% in anodic wastewater treatment. This research provides a sustainable and efficient technique for lignin valorization and wastewater treatment.

An integrated strategy for characterization of chemical constituents in Stephania tetrandra using LC–QTOF–MS/MS and the target isolation of two new biflavonoids

LC–MS has been a widely used analytical technique for identification of natural compounds. However, sophisticated and laborious data analysis is required to identify chemical components, especially new compounds, from a large LC–MS dataset. The aim of this study is to develop an integrated data-mining strategy that combines molecular networking (MN), in-house polygonal mass defect filtering (MDF), and diagnostic fragment ion filtering (DFIF) to identify phytochemicals in Stephania tetrandra based on LC–MS data. S. tetrandra samples were prepared by matrix solid-phase dispersion extraction methods and then raw MS spectra were acquired using LC–QTOF–MS/MS. MN and in-house polygonal MDF classified the compounds roughly. Modified DFIF were then used in succession to place each spectrum into a specific class. Finally, the exact structures were deduced by fragmentation pathways and related botanical biogenesis, with the help of the narrowed classification from MN and MDF. The total workflow was a combination of data filtering and identification methods for rapid characterization of known compounds (dereplication) and discovery of new compounds. Consequently, 144 compounds were identified or tentatively identified in the aerial parts and roots of S. tetrandra, including 11 potentially new compounds and 63 compounds first identified in this species. Among 144 compounds, 61 were from the aerial parts exclusively, 8 were from the roots exclusively, and 75 were found in both parts. Furthermore, two new biflavonoids were isolated with the guide of LC–MS analysis and structurally elucidated by spectroscopic methods. In conclusion, the proposed data-mining strategy based on LC–MS can be used to profile chemical constituents with high efficiency and guide the isolation of new compounds from medicinal plants. The comparison of the components of the aerial parts and roots of S. tetrandra would be helpful for the rational utilization of the medicinal plant.

False-Positive Amphetamines in Urine Drug Screens: A 6-Year Review.

Immunoassays are routinely used to provide rapid urine drug screening results in the clinical setting. These screening tests are prone to false-positive results and ideally require confirmation by mass spectrometry. In this study, we have examined a large number of urine specimens where drugs other than amphetamines may have caused a false-positive amphetamine immunoassay screening result. Urine drug screens (12,250) in a clinical laboratory that used the CEDIA amphetamine/ecstasy method were reviewed for false-positive results over a 6-year period (2015-2020). An additional 3,486 referred samples, for which confirmatory--mass spectrometry was requested, were also reviewed. About 86 in-house samples and 175 referral samples that were CEDIA false-positive screens were further analyzed by an LC-QTOF general unknown screen. Potential cross-reacting drugs were identified, and their molecular similarities to the CEDIA targets were determined. Commercial standards were also analyzed for cross-reactivity in the amphetamine/ecstasy CEDIA screen. Positive amphetamine results in 3.9% of in-house samples and 9.9% of referred tests for confirmatory analysis were false positive for amphetamines. Of these false-positive specimens, on average, 6.8 drugs were detected by the LC-QTOF screen. Several drugs were identified as possible cross-reacting drugs to the CEDIA amphetamine/ecstasy assay. Maximum common substructure scores for 70 potential cross-reacting compounds were calculated. This was not helpful in identifying cross-reacting drugs. False-positive amphetamine screens make up to 3.9-9.9% of positive amphetamine screens in the clinical laboratory. Knowledge of cross-reacting drugs may be helpful when mass spectrometry testing is unavailable.

Metabolomic profiling of bovine leucocytes transformed by Theileria annulata under BW720c treatment

BackgroundWhen Theileria annulata infects host cells, it undertakes unlimited proliferation as tumor cells. Although the transformed cells will recover their limited reproductive characteristics and enter the apoptosis process after treatment with buparvaquone (BW720c), the metabolites and metabolic pathways involved are not clear.MethodsThe transformed cells of T. annulata were used as experimental materials, and the buparvaquone treatment group and DMSO control group were used. Qualitative and quantitative analysis was undertaken of 36 cell samples based on the LC–QTOF platform in positive and negative ion modes. The metabolites of the cell samples after 72 h of drug treatment were analyzed, as were the different metabolites and metabolic pathways involved in the BW720c treatment. Finally, the differential metabolites and metabolic pathways in the transformed cells were found.ResultsA total of 1425 metabolites were detected in the negative ion mode and 1298 metabolites were detected in the positive ion mode. After drug treatment for 24 h, 48 h, and 72 h, there were 56, 162, and 243 differential metabolites in negative ion mode, and 35, 121, and 177 differential metabolites in positive ion mode, respectively. These differential metabolites are mainly concentrated on various essential amino acids.ConclusionBW720c treatment induces metabolic disturbances in T. annulata-infected cells by regulating the metabolism of leucine, arginine, and l-carnitine, and induces host cell apoptosis.Graphical abstract

Combination of high-performance thin-layer chromatography and liquid chromatography–quadrupole time-of-flight–tandem mass spectrometry analysis: a promising analytical tool for discrimination between oleo-gum resin of raw and purified Commiphora wightii

Guggulu (Commiphora wightii oleo-gum resin) is exported in more than 42 countries including developed countries like the United Kingdom and the United States of America. The demand for guggulu is more than its production. Various purification processes have been mentioned in Ayurvedic classical literature which are not explored well yet, so it is impossible to discriminate between purified and raw guggulu. In the present study, an effort was made where guggulu was purified by using cow urine and changes were analyzed by using high-performance thin-layer chromatography (HPTLC) and liquid chromatography–quadrupole time-of-flight–tandem mass spectrometry (LC–QTOF–MS/MS). In HPTLC analysis, three new bands (RF 0.31, 0.68, 0.74) and twelve other bands were observed including the bands of marker compounds guggulsterone E (RF 0.81) and Z (RF 0.85) at 254 nm. In LC–QTOF–MS/MS analysis, two new peaks at Rt 5.00 and 16.21 min were observed in cow urine-purified guggulu. After purification, all the peaks were resolute well which shows the effects of purification. Overall, in LC–MS/MS study, metabolites, viz., 5-(13′Z-nonadecenyl)resorcinol (Rt 8.31), 8β-hydroxy-3,20-dioxopregn-4,6-diene (Rt 10.33), guggulsterone E (Rt 12.99), guggulsterone Z (Rt 13.28), guggulsterol I (Rt = 15.19), mangiferolic acid (Rt = 15.27), 20(S),21-epoxy-3-oxocholest-4-ene (Rt = 15.75), and guggulsterol II (Rt 16.23) were identified. The present study reports the first time a comparative analysis of raw guggulu and cow urine-purified guggulu by using advanced analytical tools like HPTLC and LC–QTOF–MS/MS. This study may be helpful in the standardization of and quality control of raw guggulu and cow urine-purified guggulu used in various formulations of guggulu.

Human metabolism of AP-238. A novel synthetic opioid spreading in the shadow of “fentalogues”

The phenomenon of new psychoactive substances is constantly evolving, with novel synthetic opioids (NSO) representing a particular threat given their sometimes extremely high potencies and their potential to produce respiratory depression. As a consequence of recently implemented legal restrictions on fentanyl analogues, a new “legal” generation of opioids, embodied by the class of cinnamylpiperazines, popped up on the illicit drug market. AP-238 was the latest NSO of this series to be notified to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and is involved in an increasing number of acute intoxications. Adverse effects associated with the abuse of NSO, their continued evolution, increased popularity and high availability constitute a serious concern and pose an imminent hazard to public health. The identification of specific markers of consumption for these NSO is essential for clinical and forensic casework and represents a new challenge for toxicology laboratories. Referring to this, we investigated the in vivo and in vitro metabolism of AP-238. For tentative identification of the main phase-I metabolites, a pooled human liver microsomes (pHLM) assay was used. Incubation was performed for 2 hours at 37 °C in triplicates, including a blank control (no pHLM) and a zero control (no reference standard). Analyses were carried out using liquid chromatography coupled to time-of-flight mass spectrometry (LC-qToF-MS). Full scan and auto MS/MS data were acquired in one run. In a second run, full scan and broadband collision-induced dissociation (bbCID) data were acquired, checking for characteristic fragment ions (e.g., m/z 117.0699) to detect unexpected metabolites. To compare the relative abundance of the metabolites, a multiple reaction monitoring (MRM) method comprising at least two ion transitions of each metabolite, was applied using liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS). Chromatographic separation was achieved on a Kinetex® C18 column (100 × 2.1 mm, 2.6 μm). Both instruments were operated in positive ion mode. Anticipated phase-I metabolites detected in the pHLM assay were confirmed in three authentic blood samples collected during postmortem examinations and tested positive for AP-238 in the Institute of Forensic Medicine of Freiburg. In total, ten AP-238 phase-I metabolites were identified by means of LC–qToF–MS in the pHLM assay and confirmed five of these by corresponding signals in the human blood samples. They mainly consisted of products of hydroxylation, oxidation, dealkylation, and combinations of these reactions. A dealkylated/monohydroxylated metabolite was detected with the highest abundance in blood. Suggested biomarkers for AP-238 consumption are generated by monohydroxylation combined with dealkylation (most abundant) and by sole hydroxylation at the cinnamyl substituent, with the latter being highly specific. Although this investigation was carried out with blood samples due to the non-availability of urine, it is likely that these metabolites are suitable markers for urine screening as well. The combination of dealkylation with (multiple) hydroxylation(s) was also observed for further minor metabolites. The exact chemical structures of these metabolites remain unclear and would require isolation and structure elucidation e.g. by nuclear magnetic resonance spectroscopy or synthesis of reference material. However, this does not preclude their use as valid biomarkers. Implementing analytical methods for the detection of NPS metabolites is often crucial to document consumption in clinical and forensic toxicology. In vitro methods like the used pHLM assay proved to be suitable tools for prediction of biomarkers for NSO intake, although they have to be confirmed by authentic samples. If urine is not available, blood can be used for this purpose.

Accumulation and biotransformation of the novel vanillin-derived pesticide, vanisulfane, in laying hens after dietary exposure

Vanisulfane is a novel plant antiviral agent with promising prospects to prevent and control viral crop diseases. However, human health risk assessment after vanisulfane exposure from animal-derived food products remains limited. To gain insight into the accumulation and biotransformation of vanisulfane in livestock, laying hens were dietary exposed to 14C-vanisulfane. Although more than 80 % of the applied dose was observed in the excreta, vanisulfane and its metabolites accumulated in tissues, especially the liver and kidney, and was found to be transferred to eggs. A total of eight metabolites associated with both phase I and phase II metabolism were identified via 14C tracing and LC–QTOF–MS. Phase I metabolism included oxidation, hydroxylation, dechlorination and demethylation, and phase II metabolism was associated with sulfonic acid and glucuronide conjugation. The high percentages of metabolites in laying hens’ tissues and organs, illustrated the active biotransformation of vanisulfane in vivo, which suggests that the marker residues of vanisulfane should consider its major metabolites. A digestive model was also used to determine the digestive fate of vanisulfane. This study improves our understanding of vanisulfane accumulation and biotransformation in laying hens, which will be helpful for risk assessments of foods derived from animals exposed to pesticides.

Occurrence and transformation of unknown organochlorines in the wastewater treatment plant using specific Fragment-Based method with LC Q-TOF MS

Wastewater treatment plants (WWTPs) are important point sources of organochlorines in surface waters. However, comprehensive molecular-level understanding of the occurrence and transformation of organochlorines in WWTPs remains elusive. In this study, a specific fragment-based screening method with SWATH of LC Q-TOF MS was established to better understand the molecular composition of organochlorines. This method effectively excludes the non-chlorinated signals and provides multi-dimensional information (e.g., retention time, precursor ion mass, product ions, and molecular formula) with one injection to identify the possible structures of organochlorines. Eighty-seven organochlorines were successfully screened in practical wastewater samples, where 8 chlorinated sulfonic acids, 4 chlorophenols, 4 chlorinated benzenediols, and 6 chlorinated benzoic acids were further (tentatively) identified. Relative abundance of organochlorines showed that their occurrence was associated with the treatment units. In particular, anaerobic biological and NaClO treatment units contributed to the formation of chlorinated by-products. Most chlorinated by-products were substituted with more chlorine atoms than organochlorines from the influent. Furthermore, the relative abundance indicated that the fate of organochlorines were related to their structures. Chlorinated benzene sulfonic acids would be removed by adsorption on activated sludge. Most chlorinated benzoic acids were refractory, but some were likely to be chlorinated during the anaerobic process. Chlorophenols and chlorinated benzenediols might undergo chlorination, dealkylation/C-O bond breakage, and bromination. Our study offers a new tool to gain molecular information on organochlorines in complex environmental samples and highlights the importance of molecular structures when evaluating the fate of organochlorines and managing effluent discharge to surrounding waters.

Secondary or Specialized Metabolites, or Natural Products: A Case Study of Untargeted LC-QTOF Auto-MS/MS Analysis.

The large structural diversity of specialized metabolites represents a substantial challenge in untargeted metabolomics. Modern LC–QTOF instruments can provide three- to four-digit numbers of auto-MS/MS spectra from sample sets. This case study utilizes twelve structurally closely related flavonol glycosides, characteristic specialized metabolites of plant tissues, some of them isomeric and isobaric, to illustrate the possibilities and limitations of their identification. This process requires specific software tools that perform peak picking and feature alignment after spectral deconvolution and facilitate molecular structure base searching with subsequent in silico fragmentation to obtain initial ideas about possible structures. The final assignment of a putative identification, so long as spectral databases are not complete enough, requires structure searches in a chemical reference database, such as SciFindern, in attempts to obtain additional information about specific product ions of a metabolite candidate or check its feasibility. The highlighted problems in this process not only apply to specialized metabolites in plants but to those occurring in other organisms as well. This case study is aimed at providing guidelines for all researchers who obtain data from such analyses but are interested in deeper information than just Venn diagrams of the feature distribution in their sample groups.

High-Throughput Determination of Major Mycotoxins with Human Health Concerns in Urine by LC-Q TOF MS and Its Application to an Exposure Study.

Human biomonitoring constitutes a suitable tool to assess exposure to toxins overcoming the disadvantages of traditional methods. Urine constitutes an accessible biological matrix in biomonitoring studies. Mycotoxins are secondary metabolites produced naturally by filamentous fungi that produce a wide range of adverse health effects. Thus, the determination of urinary mycotoxin levels is a useful tool for assessing the individual exposure to these food contaminants. In this study, a suitable methodology has been developed to evaluate the presence of aflatoxin B2 (AFB2), aflatoxin (AFG2), ochratoxin A (OTA), ochratoxin B (OTB), zearalenone (ZEA), and α-zearalenol (α-ZOL) in urine samples as exposure biomarkers. For this purpose, different extraction procedures, namely, the Solid Phase Extraction (SPE); Dispersive Liquid–Liquid Microextraction (DLLME); and Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) methods were assessed, followed by Liquid Chromatography coupled to Quadrupole Time of Flight Mass Spectrometry with Electrospray Ionization (LC-ESI-QTOF-MS) determination. Then, the proposed methodology was applied to determine mycotoxin concentrations in 56 human urine samples from volunteers and to estimate the potential risk of exposure. The results obtained revealed that 55% of human urine samples analyzed resulted positive for at least one mycotoxin. Among all studied mycotoxins, only AFB2, AFG2, and OTB were detected with incidences of 32, 41, and 9%, respectively, and levels in the range from <LOQ to 69.42 µg/L. Risk assessment revealed a potential health risk, obtaining MoE values < 10,000. However, it should be highlighted that few samples were contaminated, and that more data about mycotoxin excretion rates and their BMDL10 values are needed for a more accurate risk assessment.

Use of Diagnostic Ions for the Detection of Fentanyl Analogs in Human Matrices by LC-QTOF.

To combat the ongoing opioid epidemic, our laboratory has developed and evaluated an approach to detect fentanyl analogs in urine and plasma by screening LC-QTOF MS/MS spectra for ions that are diagnostic of the core fentanyl structure. MS/MS data from a training set of 142 fentanyl analogs were used to select the four product ions and six neutral losses that together provided the most complete coverage (97.2%) of the training set compounds. Furthermore, using the diagnostic ion screen against a set of 49 fentanyl analogs not in the training set resulted in 95.9% coverage of those compounds. With this approach, lower reportable limits for fentanyl and a subset of fentanyl-related compounds range from 0.25 to 2.5 ng/mL in urine and 0.5 to 5.0 ng/mL in plasma. This innovative processing method was applied to evaluate simulated exposure samples of remifentanil and carfentanil in water and their metabolites remifentanil acid and norcarfentanil in urine. This flexible approach enables a way to detect emerging fentanyl analogs in clinical samples.

Qualitative LC-Q-TOF Analysis of Umbilical Cord Tissue via Data-Dependent Acquisition as an Indicator of In Utero Exposure to Toxic Adulterating Substances.

Toxic adulterants are drug or chemical agents used to add bulk volume to traditional drugs of abuse such as cocaine and heroin. These cutting agents include levamisole, metamizole, noxiptillin, phenacetin and xylazine as well as common legal drugs such as acetaminophen, caffeine, diphenhydramine, lidocaine, quinine, quetiapine and tramadol. Because they possess pharmacological activity they result in exposure of the user, but also in the case of pregnant women, the developing fetus, to potential drug toxicity. We describe the development, validation and implementation of a rapid (48 second sample-to-sample) test based on a qualitative data-dependent liquid chromatography-quadrupole time of flight mass spectrometry method for the analysis of toxic adulterating substances in umbilical cord tissue (UCT) samples. The method provides a means of studying potential in utero exposure to these agents. Library spectra comparison at three different collision energies was used in conjunction with retention time and accurate mass to identify these substances in UCT. Analytically based reporting limits were established to determine positivity rates of adulterants in UCT utilizing a standard addition approach. The method was applied to authentic cocaine and opioid positive UCT to screen for toxic adulterants. There were a total of 82 potential adulterant positives found in a 30-sample cohort of authentic UCT samples, with an average of 2.7 substances per case. Lidocaine was the predominant finding followed by caffeine, and diphenhydramine all of which could result from non-illicit drug exposure, however, there were positives for levamisole, phenacetin, noxiptillin and xylazine none of which are approved in the United States for human therapeutic use. This initial set of data established a preliminary positivity rate of potentially toxic adulterants in UCT samples positive for cocaine or opioid use.

Ganoderma spore powder contains little triterpenoids

BackgroundGanoderma spore is a minuscule germ cell ejected from Ganoderma gills during its growth maturity period, it has been considered with high exploitable potential in health-care products manufacture.MethodsAfter testing sporoderm-broken rate, the triterpenoids in 12 batches of broken and unbroken Ganoderma spore powder (GSP) samples were compared with Ganoderma lucidum fruiting body (GL) by high performance thin-layer chromatography (HPTLC) and further verified by liquid chromatography coupled with electrospray ionization quadrupole-time-of-flight mass spectrometry (LC–QTOF–MS); meanwhile, the dissolution of triterpenoids after bionic extraction was also investigated by HPTLC.ResultsThe sporoderm-broken rate of all the broken GSP samples was over 85%. The relative peak area of triterpenoids in GSP samples were lower than 50% of that in fruiting body, and the dissolution of triterpenoids in artificial gastrointestinal fluid was lower than in methanol.ConclusionsThis study demonstrated that there were little triterpenoids in GSP. Triterpenoids in GSP also seldom be dissolved in artificial gastrointestinal fluid.

Chemical Profile Analysis of Ling-Gui-Zhu-Gan Decoction by LC–QTOF MS and Simultaneous Determination of Nine Major Components Using QAMS Method

In this study, the chemical profile of Ling-Gui-Zhu-Gan Decoction (LGZGD) which was prepared by the method recorded in Synopsis of Golden Chamber was analyzed by LC–QTOF MS. A total of 89 compounds were identified, including flavonoids, pentacyclic triterpenes, glycosides, organic acids and others. Among them, nine major active components with high response value in chromatogram were selected for quantification, including liquiritin apioside, liquiritin, isoliquiritin apioside, isoliquiritin, coumarin, liquiritigenin, cinnamic acid, cinnamaldehyde and glycyrrhizic acid. A quantitative analysis of multi-components by single marker (QAMS) was applied to simultaneous determination of nine major components in LGZGD. Using liquiritin as the internal reference, the relative correction factors (RCFs) were calculated to determine the contents of other eight compounds. A series of methodological study and robustness study were carried out, which proved that the RCFs between liquiritin and others were stable. In addition, compared with the results calculated by external standard method (EMS), there were no remarkable differences between these two methods with absolute relative errors less than 3.12%. The developed QAMS method was successfully applied to preparation process analysis. In conclusion, this QAMS method was convenient, accurate and feasible enough for evaluating the quality of LGZGD.

Comparative Lipidomic Study of Human Milk from Different Lactation Stages and Milk Formulas.

In this report, we present a detailed comparison of the lipid composition of human milk (HM) and formula milk (FM) targeting different lactation stages and infant age range. We studied HM samples collected from 26 Polish mothers from colostrum to 19 months of lactation, along with FM from seven brands available on the Polish market (infant formula, follow-on formula and growing-up formula). Lipid extracts were analysed using liquid chromatography coupled to high-resolution mass spectrometry (LC–Q-TOF–MS). We found that the lipid composition of FM deviates significantly from the HM lipid profile in terms of qualitative and quantitative differences. FM had contrasting lipid profiles mostly across brands and accordingly to the type of fat added but not specific to the target age range. The individual differences were dominant in HM; however, differences according to the lactation stage were also observed, especially between colostrum and HM collected in other lactation stages. Biologically and nutritionally important lipids, such as long-chain polyunsaturated fatty acids (LC-PUFAs) containing lipid species, sphingomyelines or ether analogues of glycerophosphoethanoloamines were detected in HM collected in all studied lactation stages. The observed differences concerned all the major HM lipid classes and highlight the importance of the detailed compositional studies of both HM and FM.

Breath carbonyl levels in a human population of seven hundred participants

Oxidative stress is associated with numerous health conditions and disorders, and aldehydes are known biomarkers of oxidative stress that can be non-invasively measured in exhaled human breath. Few studies report breath aldehyde levels in human populations, and none claim participant numbers in the hundreds or more. Further, the breath community must first define the existing aldehyde concentration variance in a normal population to understand when these levels are significantly perturbed by exogenous stressors or health conditions. In this study, we collected breath samples from 692 participants and quantified C4–C10 straight chain aldehyde levels. C9 aldehyde was the most abundant in breath, followed by C6. C4 and C5 appear to have bimodal distributions. Post hoc, we mined our dataset for other breath carbonyls captured by our assay, which involves elution of breath samples onto a solid phase extraction cartridge, derivatization and liquid chromatography–quadrupole time of flight mass spectrometry (LC–qTOF). We found a total of 21 additional derivatized compounds. Using self-reported demographic factors from our participants, we found no correlation between these breath carbonyls and age, gender, body mass index (BMI), ethnicity or smoking habit (tobacco and marijuana). This work was preceded by a small confounders study, which was intended to refine our breath collection procedure. We found that breath aldehyde levels can be affected by participants’ using scented hygiene products such as lotions and mouthwashes, while collecting consecutive breath samples, rinsing the mouth with water, and filtering inspired air did not have an effect. Using these parameters to guide our sampling, subjects were instructed to avoid the prior conditions to provide a breath sample for our study.

Characterization of two unknown impurities in roxithromycin by 2D LC–QTOF/MS/MS and NMR

Two unknown impurities in roxithromycin were discovered and preliminarily characterized by two-dimensional liquid chromatography coupled with QTOF mass analyzer (2D LC–QTOF MS/MS). The column-switching technique of 2D LC made the chromatographic conditions in official standard of roxithromycin compatible with mass spectrometric detector. The complete MS/MS fragmentation patterns of the impurities were studied to obtain structural information of these impurities. Furthermore, these two impurities were separated and purified by preparative HPLC, and their structures were confirmed by 1D and 2D nuclear magnetic resonance (NMR). Structural elucidation of two impurities by 1H NMR, 13C NMR, the 1H–1H COSY, HSQC and HMBC NMR spectra has been discussed. Based on high resolution MS/MS and NMR data, the structures of these two impurities were elucidated respectively as 11-O-[(2-Methoxyethoxy) methyl] roxithromycin and de(N-methyl)-N-formyl roxithromycin. In addition, the mechanisms for formation of the impurities were also proposed.