Hybrid Triple Quadrupole-linear Ion Trap Research Articles

Single-Base Methylome Analysis of Sweet Cherry (Prunus avium L.) on Dwarfing Rootstocks Reveals Epigenomic Differences Associated with Scion Dwarfing Conferred by Grafting

Plant grafting using dwarfing rootstocks is one of the important cultivation measures in the sweet cherry (Prunus avium) industry. In this work, we aimed to explore the effects of the dwarfing rootstock “Pd1” (Prunus tomentosa) on sweet cherry ‘Shuguang2’ scions by performing morphological observations using the paraffin slice technique, detecting GA (gibberellin) and IAA (auxin) contents using UPLC-QTRAP-MS (ultra-performance liquid chromatography coupled with a hybrid triple quadrupole-linear ion trap mass spectrometer), and implementing integration analyses of the epigenome and transcriptome using whole-genome bisulfite sequencing and transcriptome sequencing. Anatomical analysis indicated that the cell division ability of the SAM (shoot apical meristem) in dwarfing plants was reduced. Pd1 rootstock significantly decreased the levels of GAs and IAA in sweet cherry scions. Methylome analysis showed that the sweet cherry genome presented 15.2~18.6%, 59.88~61.55%, 28.09~33.78%, and 2.99~5.28% methylation at total C, CG, CHG, and CHH sites, respectively. Shoot tips from dwarfing plants exhibited a hypermethylated pattern mostly due to increased CHH methylation, while leaves exhibited a hypomethylated pattern. According to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, DMGs (differentially methylated genes) and DEGs (differentially expressed genes) were enriched in hormone-related GO terms and KEGG pathways. Global correlation analysis between methylation and transcription revealed that mCpG in the gene body region enhanced gene expression and mCHH in the region near the TSS (transcription start site) was positively correlated with gene expression. Next, we found some hormone-related genes and TFs with significant changes in methylation and transcription, including SAURs, ARF, GA2ox, ABS1, bZIP, MYB, and NAC. This study presents a methylome map of the sweet cherry genome, revealed widespread DNA methylation alterations in scions caused by dwarfing rootstock, and obtained abundant genes with methylation and transcription alterations that are potentially involved in rootstock-induced growth changes in sweet cherry scions. Our findings can lay a good basis for further epigenetic studies on sweet cherry dwarfing and provide valuable new insight into understanding rootstock–scion interactions.

Metabolite identification of salvianolic acid A in rat using post collision-induced dissociation energy-resolved mass spectrometry

BackgroundAs one of the most famous natural products, salvianolic acid A (SAA) is undergoing clinical trials for the treatments of angina pectoris and coronary heart disorders. However, the in vivo metabolites of SAA have only been tentatively identified, leading to a barrier for precise therapeutical drug monitoring.MethodsUltra-high performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry (UPLC–Qtof-MS/MS) was firstly employed to acquire high-resolution MS1 and MS2 spectra for all metabolites. Through paying special attention onto the features of ester bond dissociation, metabolism sites were restricted at certain regions. To further determine the metabolism site, such as the monomethylated products (M23, M25, and M26), post collision-induced dissociation energy-resolved mass spectrometry (post-CID ER-MS) was proposed through programming progressive exciting energies to the second collision chamber of hybrid triple quadrupole-linear ion trap mass spectrometry (Qtrap-MS) device.ResultsAfter SAA oral administration, 29 metabolites (M1–M29), including five, thirteen, and sixteen ones in rat plasma, urine, and feces, respectively, were detected in rats. The metabolism route was initially determined by applying well-defined mass fragmentation pathways to those HR-m/z values of precursor and fragment ions. Metabolism site was limited to SAF- or DSS-unit based on the fragmentation patterns of ester functional group. Through matching the dissociation trajectories of concerned 1st-generation fragment ions with expected decomposition product anions using post-CID ER-MS strategy, M23 and M25 were unequivocally assigned as 3'-methyl-SAA and 3''-methyl-SAA, and M26 was identified as 2-methyl-SAA or 3-methyl-SAA. Hydrolysis, methylation, glucuronidation, sulfation, and oxidation were the primary metabolism channels being responsible for the metabolites' generation.ConclusionTogether, the metabolism regions and sites of SAA metabolites were sequentially identified based on the ester bond dissociation features and post-CID ER-MS strategy. Importantly, the present study provided a promising way to elevate the structural identification confidence of natural products and metabolites.Graphical abstract

Floating plastics as integrative samplers of organic contaminants of legacy and emerging concern from Western Mediterranean coastal areas

This study investigates the role of floating plastics as integrative samplers of organic contaminants. To this end, plastics items were collected in two Western Mediterranean coastal areas: the Mar Menor lagoon, and the last transect of Ebro river. Floating plastics were identified and characterized by attenuated total reflection Fourier-transform infrared spectrometry. Then, organic contaminants were extracted from plastic items by ultrasonic extraction with methanol, and the concentrations of 168 regulated and emerging contaminants were analysed. These compounds were analysed by stir bar sorptive extraction coupled to gas chromatography–mass spectrometry (GC–MS), except for bisphenol analogues, which were analysed with a ultraperformance liquid chromatography pump coupled to a triple quadrupole mass spectrometer (UHPLC-MS/MS), and pharmaceutical compounds, determined by UPLC coupled to hybrid triple quadrupole-linear ion trap mass spectrometer (UPLC-MS/MS). All the contaminants groups considered were detected in the samples, being particularly relevant the contribution of plastic additives. The most frequently detected contaminants were UV-filters, PAHs, pharmaceuticals and synthetic musks. Apart from plasticizers, the individual contaminants octocrylene, homosalate, galaxolide, salycilic acid and ketoprofen were frequently detected in plastics items. The results pointed out to urban and touristic activities as the main sources of pollution in the coastal areas investigated. The utility of floating plastics as integrative samplers for the detection of organic contaminants in aquatic ecosystems has been demonstrated.

Eco-friendly simultaneous extraction of pectins and phenolics from passion fruit (Passiflora edulis Sims) peel: Process optimization, physicochemical properties, and antioxidant activity

The objective of this study was to simultaneously extract passion fruit (Passiflora edulis) peel pectins and phenolics using deep eutectic solvents, to evaluate their physicochemical properties and antioxidant activity. By taking L-proline: citric acid (Pro-CA) as the optimal solvent, the effect of extraction parameters on the yields of extracted passion fruit peel pectins (PFPP) and total phenolic content (TPC) was explored by response surfaces methodology (RSM). A maximum pectin yield (22.63%) and the highest TPC (9.68 mg GAE/g DW) were attained under 90 °C, extraction solvent pH = 2, extraction time of 120 min and L/S ratio of 20 mL/g. In addition, Pro-CA-extracted pectins (Pro-CA-PFPP) and HCl-extracted pectins (HCl-PFPP) were subjected to high performance gel permeation chromatography (HPGPC), Fourier transform infrared spectroscopy (FT-IR), thermogram analysis (TG/DTG) and rheological measurements. Results verified that the Mw and thermal stability of Pro-CA-PFPP were higher than those of HCl-PFPP. The PFPP solutions featured a non-Newtonian behavior, and compared with commercially pectin solution, PFPP solution exhibited a stronger antioxidant activity. Additionally, passion fruit peel extract (PFPE) exhibited stronger antioxidant effects than PFPP. The results of ultra-performance liquid chromatography hybrid triple quadrupole-linear ion trap mass spectrometry (UPLC-Qtrap-MS) and high performance liquid chromatography (HPLC) analysis showed that (−)-epigallocatechin, gallic acid, epicatechin, kaempferol-3-O-rutin and myricetin were the main phenolic compounds in PFPE and PFPP. Our results suggest that Pro-CA can be considered as an eco-friendly solvent for high-efficient extraction of high-value compounds from agricultural by-products.

LC-MS-based metabolite profiling of aqueous extract of Pergularia tomentosa L. and its anti-hyperglycemic effect.

In this study, to find scientific evidence for the traditional use of Pergularia tomentosa as an anti-diabetic remedy, the effects of its aqueous extract on streptozotocin-induced diabetes mellitus in rats were evaluated. Wistar rats were fasted overnight and diabetes mellitus was induced using streptozotocin (50 mg/kg body weight). The rats were randomly and equally divided into four groups (n=5): group I (normoglycaemic control), group II (diabetic rats), group III (diabetic rats treated with 200 mg/kg BW of an aqueous extract of P. tomentosa), group IV (normoglycemic rats treated with 200 mg/kg BW of an aqueous extract of P. tomentosa). Chemical profiling of the aqueous extract was carried out using liquid chromatography coupled with electrospray ionization and multiple-stage linear ion-trap and orbitrap high-resolution mass spectrometry (LC-ESI/LTQOrbitrap/MS/MS). In addition, the quantitative determination of the main cardenolides in the extract was carried out by an analytical approach based on LC coupled to tandem mass spectrometry with ESI source and hybrid triple quadrupole-linear ion trap mass analyzer (LC-ESI/QTrap/MS/MS). Aqueous extract of P. tomentosa showed a reasonable reduction in blood glucose level. Probably, the P. tomentosa effect on hyperglycemic and hyperlipidemic diabetic animals was associated with antioxidant properties, triglyceride levels, as well as liver enzymes. Meanwhile, LC-ESI/LTQOrbitrap/MS/MS analysis led to identification of double-linked cardenolides along with cardenolides and flavone glycosides as the main bioactive compounds. The extract decreased the glucose level and induced a beneficial effect on the lipid profile, probably due to the presence of cardenolide glycosides.

Development and Validation of a Highly Sensitive and Rapid LC-MS3 Strategy to Determine Oxcarbazepine and Its Active Metabolite in the Serum of Patients with Epilepsy and Its Application in Therapeutic Drug Monitoring

A sensitive and rapid bioanalytical method based on the LC-triple-stage fragmentation (LC-MS3) strategy on a hybrid triple quadrupole-linear ion trap mass spectrometer in combination with protein precipitation extraction for sample pretreatment has been developed and validated for the simultaneous determination of the antiepileptic drug oxcarbazepine (OXC) and its main active metabolite (MHD) in human serum. The separation was performed on a Waters XBridge BEH C18 column (2.5 µm, 2.1 × 50 mm) in isocratic elution with 0.1% formic acid in water and methanol (50:50, v:v) as the mobile phase. The run time for each sample was 2.0 min. The calibration curves ranging from 25 to 1600 ng/mL for OXC and from 0.5 to 32 μg/mL for MHD showed correlation coefficients (r) better than 0.99. All of the validation data, such as precision, accuracy and other parameters, fit the requirements of the current bioanalytical method validation guidelines. The LC-MS3 method for quantitation of OXC and MHD was compared with the LC-MRM based method. Passing–Bablok regression coefficients and Bland–Altman plots showed that the developed LC–MS3 method is a reliable method for quantitative analysis of OXC and MHD. The proposed LC-MS3 method was successfully applied to determine the serum concentrations of OXC and MHD to support a clinical study.

Changes in Phospholipid Composition of the Human Cerebellum and Motor Cortex during Normal Ageing.

(1) Background: Changes in phospholipid (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, i.e., PC, PE and PS) composition with age in the mitochondrial and microsomal membranes of the human cerebellum and motor cortex were examined and compared to previous analyses of the prefrontal cortex, hippocampus and entorhinal cortex. (2) Methods: Nano-electrospray ionization on a hybrid triple quadrupole–linear ion trap mass spectrometer was used to analyse the brain regions of subjects aged 18–104 years. (3) Results: With age, the cerebellum showed many changes in the major phospholipids (>10% of the phospholipid class). In both membrane types, these included increases in PE 18:0_22:6 and PS 18:0_22:6, decreases in PE 18:0_20:4 and PS 18:0_18:1 and an increase in PC 16:0_16:0 (microsomal membrane only). In addition, twenty-one minor phospholipids also changed. In the motor cortex, only ten minor phospholipids changed with age. With age, the acyl composition of the membranes in the cerebellum increased in docosahexaenoic acid (22:6) and decreased in the arachidonic (20:4) and adrenic (22:4) acids. A comparison of phospholipid changes in the cerebellum, motor cortex and other brain areas is provided. (4) Conclusions: The cerebellum is exceptional in the large number of major phospholipids that undergo changes (with consequential changes in acyl composition) with age, whereas the motor cortex is highly resistant to change.

Advantages of MS/MS/MS (MRM3) vs classic MRM quantification for complex environmental matrices: Analysis of beta-lactams in WWTP sludge

When dealing with complex matrices such as wastewater treatment plant (WWTP) sludge or animal manure, usual MRM quantification may lack enough sensitivity or accuracy due to the presence of numerous interfering compounds co-extracted from the matrix. To circumvent the sensitivity and specificity loss, the method development can be focused on sample extraction, purification or/and optimisation of the detection. In this study, we propose an enhancement of a method for the analysis of five beta-lactams (Amoxicillin, Ampicillin, Cefapirin, Ceftiofur, and Cloxacillin) in WWTP, with the use of a hybrid triple quadrupole-Linear Ion Trap (LIT) spectrometer, enabling triple stage MS acquisition, namely MRM3, in place of the usual MS/MS detection. The adaptation of various parameters such as the secondary fragmentation energy, excitation, and accumulation times of the secondly generated ion are described. The method was then validated and enabled quantification limits between 0.8 and 14.7 ng g−1 associated with accurate quantification (between 98% and 113%). This method is the first to report the use of MRM3 acquisition in an environmental matrix as complex as sludge.

Characterization of drug-related impurities using UPLC-QTrap-MS/MS in three scan modes: Application to daptomycin

A new non-targeted screening strategy was developed for the detection and relative quantification of known and potentially unknown drug-related impurities in the active pharmaceutical ingredient daptomycin using ultra-high performance liquid chromatography, coupled with hybrid triple quadrupole-linear ion trap electrospray ionization mass spectrometry in three different modes. The precursor ion scan mode was first applied using the m/z transitions of 159.2, 313.2, and 341.2 for rapid screening of all daptomycin-related impurities. Precursor ions detected at specific retention times were selected, fragmented, and focused in enhanced product ion scan mode to obtain secondary ion mass spectra and identify drug-related impurities. Finally, each potential compound was relatively quantified in multiple reaction monitoring mode. Repeatability experiments suggest that this impurity profiling method is a reliable strategy for identifying unknown impurities in daptomycin and other drugs.

Metabolite Profiling and Characterization of LW6, a Novel HIF-1α Inhibitor, as an Antitumor Drug Candidate in Mice.

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.

Identification of metabolites in rat plasma,bile,urine and feces after oral administration of Cinnamomi Cortex aqueous extract by UPLC-Qtrap-MS

An ultra-performance liquid chromatography hybrid triple quadrupole-linear ion trap mass spectrometry(UPLC-QtrapMS) method was established to identify the metabolites in rat plasma,bile,urine and feces after oral administration of Cinnamomi Cortex(CC) aqueous extract. Several survey experiments,such as enhanced mass spectrum scan(EMS),precursor ion scan(PI),neutral loss scan(NL) and multiple ions monitoring(MIM) were applied to search target components,and two separate enhanced product ion(EPI) scans were triggered via information-dependent acquisition(IDA) method to generate the MS/MS spectra. According to the mass spectrometric data collected from reference standards and reported literature,the structures of metabolites were deduced. A total of76 metabolites and 5 original compounds were tentatively identified in rats after oral administration of CC aqueous extract. Deglycosylation,methylation,sulfonation,and glucuronidation were observed as the primary metabolic pathways for the chemical constituents of CC. These data are able to benefit the clarification of the therapeutic material basis,the clinical usage and further R&D of CC.

Comparative study on metabolic profiling and excretion in rat bile between combination of notoginseng total saponins and safflower total flavonoids and its individual extracts by LC–MS/MS

The combination of notoginseng total saponins (NS) and safflower total flavonoids (SF), namely CNS, presents a synergistic protection effect on the myocardial ischemia rats. The aim of this study was to find the clues for their synergistic actions by comparing the biliary metabolism and excretion profiles after oral administration of CNS and its individual extracts. An ultra-performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometer (UPLC-QTRAP-MS/MS) platform was used to identify and quantify the CNS-derived components in bile. The neutral losses, precursor ions, and predictive multiple reaction monitoring (pMRM) scans were firstly used to detect the CNS-derived ingredients in vivo. A total of 43 components, including 38 flavonoids and 5 ginsenosides were tentatively identified according to the previously established chemical and metabolic profiles of NS and SF. Afterwards, the primary circulating and biological components, hydroxysafflor yellow A (HSYA), ginsenosides Rg1 (GRg1), Re (GRe), and Rd (GRd) were chosen to compare the bile excretion between CNS and its individual extract groups, by using a validated LC-MRM-MS/MS method. The approach was proved to be well satisfied the related requirements from the guidelines of FDA (specificity, calibration curve, sensitivity, precision, accuracy, matrix effect, recovery, and stability). Comparing with the SF and NS groups, the combination group did not affect the metabolic pathways of the CNS-related components, however, it decreased the cumulative excretion ratios of HSYA, GRg1, GRe, and GRd. In conclusion, the compatibility of SF and NS could reduce the bile excretion of the CNS-derived compounds, which may be one of the reasons for the enhancement of anti-myocardial ischemia after combination.

Optimal collision energy is an eligible molecular descriptor to boost structural annotation: An application for chlorogenic acid derivatives-focused chemical profiling

Tandem mass spectrometry (MS/MS), in particular high-resolution MS/MS, is able to provide element compositions and substructures for the detected signals. However, it is still challenging to configure the whole structures via linking those substructures. Efforts were devoted here to propose and validate optimal collision energy (OCE) to be an auxiliary structural clue to mass-to-charge ratios (m/z), and online energy-resolved MS was developed to yield OCEs. Chlorogenic acid derivatives (CADs) were utilized as the proof-of-concept because diverse isomers usually initiated by the different linkage manners between the quinic acid/shikimic acid and cinnamoyl substituents(s), i.e. caffeoyl group, coumaroyl group, etc. Liquid chromatography–hybrid ion trap-time of flight MS (LC–IT-TOF–MS) was implemented to capture CADs in two well-known herbal medicines namely Lonicerae japonicae Flos and Inulae Flos. Afterwards, hybrid triple quadrupole-linear ion trap MS (Qtrap-MS) was deployed to acquire OCEs for the primary fragmentation pathways of all detected CADs through online energy-resolved MS. On the other side, structural calculations were conducted to figure out the relationships between OCEs and bond properties. Isomeric differences occurred for OCEs, and LC elution program as well as ionization parameters could not affect OCEs. Twenty-four and thirty-one CADs were hunted and putatively identified by LC–IT-TOF-MS in Lonicerae japonicae Flos and Inulae Flos, respectively, and the structural annotation was advanced by applying the OCE-bond property relationships. To verify the structures, CADs-of-interest were purified from Lonicerae japonicae Flos using an automated fraction collector and definitely identified with NMR spectroscopy. Exact consistence occurred for the structural identification of mono-caffeoylquinic acid isomers between LC–MS/MS and NMR analyses. Consequently, OCE is an inherent physicochemical parameter of a given compound and is an eligible structural descriptor to offset the ability of LC–MS/MS towards the chemical profiling of complex matrices.

Serial hyphenation of dried spot, reversed phase liquid chromatography, hydrophilic interaction liquid chromatography, and tandem mass spectrometry towards direct chemical profiling of herbal medicine-derived liquid matrices, an application in Cistanche sinensis

Inspired by dried blood spots (DBS), “dried spots of herbal medicines” (DSHM) concept was proposed here. In response to this superior sampling means, a new platform integrating dried spot, serially coupled reversed phase liquid chromatography and hydrophilic interaction liquid chromatography (RPLC-HILIC), and tandem mass spectrometry (MS/MS), was configured for directly, comprehensively chemical profiling of HM-derived liquid matrices. As an important original source of Cistanches Herba (Chinese name: Roucongrong) that is a well-known tonic HM, Cistanche sinensis (Csi) was employed to illustrate and validate the applicability. Dried spots (I.D. 3.0 mm) were prepared by loading 2 μL aliquots of Csi extract onto filter paper. Each dried spot was packed into an in-line filter holder (I.D. 3.0 mm × 4.0 mm) and then inserted behind the auto-sampler of a well-defined instrumentation named RPLC-HILIC–MS/MS. Hybrid ion trap-time of flight MS and hybrid triple quadrupole-linear ion trap MS were deployed in combination for the in-depth MS/MS data acquisition of diverse chemical families, such as phenylethanoid glycosides, lignans, iridoids, amino acids, and so forth. To assist chemical profiling, an in-house chemical library was built by collecting as much prior knowledge as possible. A total of 88 components were detected and tentatively annotated in Csi by matching their multi-stage MS spectra with those of authentic standards and literature data. Collectively, DSHM carried all merits of DBS, and the integrative dried spot-RPLC-HILIC-MS/MS platform was a promising analytical tool for direct chemical analysis and rapid quality evaluation of HMs, in particular those traditional Chinese medicine injections.

Identification of forced degradation products of lacosamide by LC-QQLIT-MS and LC-IT/TOF-MS

Lacosamide, a new type of antiepileptic drug, was subjected to forced degradation under the conditions of hydrolysis (acidic and alkaline), oxidation, dry heat, and photolysis to characterize its possible degradation products. The drug showed significant degradation under acidic, alkaline and oxidative conditions. The degradation products were separated on an Agilent Zorbax SB-C18 column with gradient elution using a mobile phase consisting of acetonitrile and ammonium acetate (0.002 mol/L) with formic acid as additive. A combination of liquid chromatography hybrid triple quadrupole-linear ion trap mass spectrometry (LC–QqLIT-MS) and liquid chromatography hybrid ion trap/time-of-flight mass spectrometry (LC-IT/TOF-MS) was used to identify degradation products. A total of 7 products including 4 novel degradation products were characterized. The mechanisms of degradation products of lacosamide were discussed. Application of the method to study degradation products of lacosamide provided fragment information, allowing further investigation of the degradation pathways and intrinsic stability of the drug.

LC-MS Analysis of Dimethyl Fumarate in Rat plasma with Measurement Uncertainty Estimation

Dimethyl fumarate (DMF) is the methyl ester of fumaric acid initially recognized as a very effective hypoxic cell radio sensitizer. Phase III clinical trials found that DMF successfully reduced relapse rate and increased time to progression of disability in multiple sclerosis. Small molecules like dimethyl fumarate pose particular difficulties when analyzing biological samples due to the increased possibility of matrix effects and in this scenario DMF immediately converts to its active metabolite MMF (Monomethyl fumarate) by oral route. It is for this reason that a sensitive liquid chromatography-tandem mass spectrometry method has now been developed for the analysis of DMF and for studying the pharmacokinetic profile in rats. Sample preparation was by rapid protein precipitation with acetonitrile. Analyte separation was achieved on a reversed-phase XTerra MS C18 column (100 x 3.9 mm, 3.5g) with 0. 01M ammonium formate and acetonitrile in gradient mode as the mobile phase at a flow rate of 1.0 mL/min and analyzed by a hybrid triple-quadrupole linear ion trap mass spectrometer in positive electrospray ionization mode for both DMF and MMF. Limits of detection, and quantification were 20 and 50 ng/mL, for DMF and 1 and 10 ng/mL for MMF respectively. Calibration curve showed excellent linearity within the 50–2500 ng/mL range for DMF and 10–500 ng/mL range (r2 > 0.999) for MMF. Intra-and inter-day precision defined by coefficient of variation was <10% and accuracy (bias %) was within 90–110%. Measurement uncertainty estimation was 8.6% for DMF and 11.6% for MMF. The method has been successfully used in the analysis of DMF and MMF in rat plasma following its administration to male wistar rats for pharmacokinetic studies.

Definitely simultaneous determination of three lignans in rat using ultra-high performance liquid chromatography-tandem mass spectrometry

Herbal medicines (HMs) have been widely demonstrated to be extremely complicated chemical pools, notably the occurrences of isomers that usually possess similar chromatographic and spectrometric behaviors, and the complexity will be further boosted in HMs-treated biological samples. Hence, there is a technical barrier regarding identity confirmation towards the acquisition of reliable quantitative information for those compounds-of-interest in biological matrices, although tandem mass spectrometry (MS/MS) is a favored tool because of the advantages in terms of specificity and selectivity, in particular being hyphenated with ultra-high performance liquid chromatography (UHPLC). More structural information should be involved in addition to retention times and precursor-to-product ion transitions, to consolidate the identities of the captured signals. Attempts were made here to strengthen quantitation confidences via matching MS2 spectra and relative response-collision energy curves (RRCECs) between each pair of suspect peak and the authentic signal, and UHPLC coupled to hybrid triple quadrupole-linear ion trap mass spectrometry (Qtrap-MS) that enabled simultaneous acquisition of qualitative and quantitative information acted as the analytical tool. Definitely simultaneous determination of a pair of regio-isomers namely 6‑hydroxy‑4‑(4‑hydroxy‑3‑methoxyphenyl)‑3‑hydroxymethyl‑7‑methoxy‑3,4‑dihydro‑2‑naphthaldehyde (VB-1) and vitedoin A (VB-2), together with another analogue namely detetrahydroconidendrin (VB-3), in rat plasma as well as tissues was conducted following oral administration of the extract of fruits of Vitex negundo var. cannabifolia. Diverse method validation assays in regard of selectivity, linearity, intra- and inter-day variations, matrix effect, and recovery were employed to demonstrate the newly developed method being able to fulfill the demands of rational quantitation. Overall, the current study offers a promising approach to accomplish confidence-enhanced quantitation in biological matrices.

Development of Ultra Performance Liquid Chromatography Tandem Mass Spectrometry Method for Simultaneous Identification and Quantitation of Potential Osteogenic Phytochemicals in Butea monosperma.

An ultra performance liquid chromatography coupled with hybrid triple-quadrupole linear ion trap tandem mass spectrometry (UPLC-ESI-QqQLIT-MS-MS) method in multiple reaction monitoring mode was developed for identification and simultaneous determination of potential osteogenic compounds in ethanol extracts of different plant parts of Butea monosperma collected from different geographical regions. The chromatographic separation was carried out on an Acquity UPLC CSH C18 column (1.7 μm, 2.1 × 100 mm) with 0.1% (v/v) formic acid in water and methanol as mobile phase under gradient conditions in 8 min. The developed method was validated according to the guidelines of international conference on harmonization. The correlation coefficients of all the calibration curves were ≥0.9995 and recoveries ranged from 95.2 to 105.8% (RSD ≤ 1.95%). Relative standard deviations of intra-day, inter-day precisions and stability were ≤1.74, 1.84 and 2.8%, respectively. The quantitative results showed remarkable differences in the content of all potential osteogenic compounds in different parts of the plant as well as samples from different geographical regions. Quantitative variations studied from principal component analysis indicated tentative markers for B. monosperma cultivars which can discriminate sample of different geographical regions.

Method development and application for multi-component quantification in rats after oral administration of Longxuetongluo Capsule by UHPLC–MS/MS

Although wide applications towards ischemic stroke in clinic, the therapeutic materials of Longxuetongluo Capsule (LTC) that is composed of total phenolic extract of Chinese dragon’s blood, are still largely unclear. Exposure pattern characterization of those drug-derived components in vivo, notably in circulation system has been recommended as a viable approach to disclose the effective components of a given herbal medicine. Herein, we aimed to develop a robust method being capable of multi-component quantification in either rat plasma or tissues following oral administration of LTC, and to clarify the kinetic profiles of 11 primary drug-derived phenolic derivatives. Proteins precipitation was carried out for the plasma as well as homogenized tissue samples with acetonitrile. Chromatographic separations were achieved using UHPLC equipped with a shim-pack XR-ODS II column, and confidence-enhanced detection was accomplished through the joint employment of selected-reaction monitoring and tandem mass spectrometry (SRM-MS/MS) on a hybrid triple quadrupole-linear ion trap mass spectrometer. Diverse validation assays proved the method to be sensitive, precise, and rapid for simultaneous determination of those 11 components. Pharmacokinetic and tissue distribution investigations were subsequently conducted in rat after a single 500 mg/kg oral dose. Rapid absorption (Tmax, 11.53–68.27 min) and elimination (T1/2, 6.893–57.90 min) occurred for all analytes-of-interest. Extensive occurrences were observed for 7,4′-dihydroxy-5-methoxyhomoisoflavanone (Cmax, 340.0 ng/mL), thevetiaflavone (Cmax, 42.86 ng/mL), 5,7,4′-trihydroxyhomoisoflavanone (Cmax, 41.55 ng/mL), and pterostilbene (Cmax, 25.49 ng/mL) in plasma. Significant distributions occurred for all analytes in the liver as well as kidney, and several compounds could be found in brain. The findings described are envisioned to provide promising information for the in-depth clarification of the therapeutic entities, and also to offer a practical approach for therapeutic drug monitoring of LTC in clinic.

Geographic impact evaluation of the quality of Alismatis Rhizoma by untargeted metabolomics and quantitative assay.

The geographic impact on the quality of Alismatis Rhizoma (derived from the tuber of Alisma orientale), a reputable diuretic traditional Chinese medicine, has seldom been evaluated. Here a metabolomics-driven approach targeting the bioactive protostane triterpenes was developed, by incorporating UHPLC with quadrupole time-of-flight mass spectrometry-based untargeted metabolite profiling and multiple reaction monitoring quantitative assay, to probe the triterpene differences between Alismatis Rhizoma samples collected from Sichuan, Fujian, and Jiangxi Provinces. Following the metabolomics workflows, the samples from Sichuan and Jiangxi displayed distinct differences in their triterpene profiles, whereas those from Fujian showed remarkable intra-class variation. Twenty-three triterpenes were identified to contribute most to the differentiated clustering. A sensitive, precise, repeatable, and accurate quantitative assay method was established on a hybrid triple quadrupole-linear ion trap mass spectrometer to quantify the contents of eight triterpene compounds. Taking into account the metabolomics and quantitation results, alisol B 23-acetate and alisol A are significantly different in Alismatis Rhizoma from Sichuan and Jiangxi Provinces, and they may have the potential for geographic discrimination. These results illustrate how geographic difference impacts the triterpene chemistry of Alismatis Rhizoma. Metabolomics-driven chemical comparison is suitable for the quality evaluation of traditional Chinese medicine.