Metabolites In Biosamples Research Articles

Determination of four amide synthetic cannabinoid isomers by ultra-high performance liquid chromatography-high resolution mass spectrometry

Isomerization commonly occurs in synthetic cannabinoids (SCs). Owing to the few differences in their structure and properties, it is difficult to simultaneously separate and identify them. Thus, the identification of synthetic cannabinoids is challenging, posing a threat to public security. This study aims to separate and identify four SCs, which are 2-[1-(5-fluoropentyl)-1H-indole-3-formylamino]-3,3-dimethylbutyrate methyl ester (5F-MDMB-PICA), 2-[1-(5-fluoropentyl)-1H-indole-3-formylamino]-3-methylbutyrate ethyl ester (5F-EMB-PICA), N-(1-amino-2,2-dimethyl-1-oxobutyl-2-yl)-1-butyl-1H-indazole-3-formamide (ADB-BINACA), N-(1-carbamoyl-2-methylpropyl)-1-pentyl indazole-3-formamide (AB-PINACA).Supercritical fluid chromatography-mass spectroscopy (SFC-MS) can realize the effective separation of some cannabinoid isomers. However, most laboratories are not equipped with SFC-MS systems. Ultra-high performance liquid chromatography-high resolution mass spectroscopy (UHPLC-HRMS) effectively combines the excellent efficient separation characteristics of liquid chromatography and the powerful qualitative ability of mass spectrometry. It is a commonly used technical method for the detection of amide synthetic cannabinoids and their metabolites in vivo and in vitro because of its advantages of high accuracy and efficiency. Liquid chromatography allows the separation of tested components by exploiting the difference in the partition coefficients between the mobile and stationary phases. When the two phases are in relative motion, the tested components are divided between the two phases, facilitating the separation and analysis of each component. Although the difference in the polarities of the tested amide synthetic cannabinoid isomeric substances is extremely small, liquid chromatography can induce a strong separation effect. The advantages of UHPLC-HRMS include high resolution imparted by mass spectrometry and high sensitivity, allowing its application in the qualitative analysis of various substances. Through UHPLC-HRMS, trace analytes at the nanogram scale as well as pure drugs and their metabolites in biosamples can be detected. This study proposed a method for the determination of two pairs of amide synthetic cannabinoid isomers-5F-EMB-PICA and 5F-MDMB-PICA, ADB-BINACA and AB-PINACA-through UHPLC-HRMS. A Hypersil GOLD C18 column (100 mm×2.1 mm, 1.9 μm) was selected for separation via liquid chromatography, and gradient elution was performed with methanol containing 0.1% formic acid and a 0.1% formic acid aqueous solution containing 10 mmol/L ammonium formate. Full scan/data-dependent secondary mass spectrometry (Full MS/dd-MS2) was conducted in the positive ion mode for detection. The results indicated that the four synthetic cannabinoid isomers could be accurately detected under the abovementioned conditions. The resolution between 5F-EMB-PICA and 5F-MDMB-PICA was 2.06, while that between ADB-BINACA and AB-PINACA was 1.22, indicating the effective separation and detection of both pairs. Furthermore, method validation was conducted to ensure the accuracy of the proposed method. The relationship of the four amide synthetic cannabinoid isomers exhibited excellent linearity. The correlation coefficients (R2) were >0.99. Moreover, the matrix effects of the four SCs in hair samples were between 88.67% and 111.76% and the recoveries were 96.23%-105.11%. The intra-day and inter-day precisions (RSDs) were <10%. The proposed method was used to identify the case materials. AB-PINACA was detected in a hair sample at a content of 0.73 μg/g. 5F-MDMB-PICA was detected in a tobacco sample at a content of 11.3 mg/g. The results indicate that the proposed method can be used for the examination of practical samples conducted by public security organizations. This study provides a reference method for the identification of synthetic cannabinoid isomers.

Diazo-carboxyl Click Derivatization Enables Sensitive Analysis of Carboxylic Acid Metabolites in Biosamples.

Carboxylic acids are central metabolites in bioenergetics, signal transduction, and post-translation protein regulation. However, the quantitative analysis of carboxylic acids as an indispensable part of metabolomics is prohibitively challenging, particularly in trace amounts of biosamples. Here we report a diazo-carboxyl/hydroxylamine-ketone double click derivatization method for the sensitive analysis of hydrophilic, low-molecular-weight carboxylic acids. In general, our method renders a 5- to 2000-fold higher response in mass spectrometry along with improved chromatographic separation. With this method, we presented the near-single-cell analysis of carboxylic acid metabolites in 10 mouse egg cells before and after fertilization. Malate, fumarate, and β-hydroxybutyrate were found to decrease after fertilization. We also monitored the isotope labeling kinetics of carboxylic acids inside adherent cells cultured in 96-well plates during drug treatment. Finally, we applied this method to plasma or serum samples (5 μL) collected from mice and humans under pathological and physiological conditions. The double click derivatization method paves a way toward single-cell metabolomics and bedside diagnostics.

Application of plasma metabolome for monitoring the effect of rivaroxaban in patients with nonvalvular atrial fibrillation.

Rivaroxaban, an oral factor Xa inhibitor, has been used to treating a series of thromboembolic disorders in clinical practice. Measurement of the anticoagulant effect of rivaroxaban is important to avoid serious bleeding events, thus ensuring the safety and efficacy of drug administration. Metabolomics could help to predict differences in the responses among patients by profiling metabolites in biosamples. In this study, plasma metabolomes before and 3 hours after rivaroxaban intake in 150 nonvalvular atrial fibrillation (NVAF) patients and 100 age/gender-matched controls were analyzed by liquid chromatography coupled with mass spectrometry (LC–MS/MS). When compared with controls, a total of thirteen plasma metabolites were differentially expressed in the NVAF patients. Pathway analysis revealed that purine and lipid metabolism were dysregulated. A panel of three metabolites (17a-ethynylestradiol, tryptophyl-glutamate and adenosine) showed good predictive ability to distinguish nonvalvular atrial fibrillation with an area under the receiver operating characteristic curve (AUC) of 1 for the discovery phase and 1 for validation. Under rivaroxaban treatment, a total of seven metabolites changed, the lipid and glycosylphosphatidylinositol biosynthesis pathways were altered and the panel consisting of avocadene, prenyl glucoside and phosphatidylethanolamine showed predictive ability with an AUC of 0.86 for the discovery dataset and 0.82 for the validation. The study showed that plasma metabolomic analyses hold the potential to differentiate nonvalvular atrial fibrillation and can help to monitor the effect of rivaroxaban anticoagulation.

Improved structural annotation of triterpene metabolites of traditional Chinese medicine in vivo based on quantitative structure-retention relationships combined with characteristic ions: Alismatis Rhizoma as an example

As a fast, sensitive and selective method, liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS) has been used for studying the in vivo metabolism of traditional Chinese medicine (TCM). However, the rapid discovery and characterization of metabolites, especially isomers, remain challenging due to their complexity and low concentration in vivo. This study proposed a strategy to improve the structural annotation of prototypes and metabolites through characteristic ions and a quantitative structure-retention relationship (QSRR) model, and Alismatis Rhizoma (AR) triterpenes were used as an example. This strategy consists of four steps. First, based on an in-house database reported previously, prototypes and metabolites in biosamples were preliminarily identified. Second, the candidate structures of prototype compounds and metabolites were determined by characteristic ions, databases or potential metabolic pathways. Then, a QSRR model was established to predict the retention times of the proposed structure. Finally, the structures of unknown prototypes and metabolites were determined by matching experimental retention times with the predicted values. The QSRR model built by the genetic algorithm-multiple linear regression (GA-MLR) has excellent regression correlation (R2 = 0.9966). Based on this strategy, a total of 118 compounds were identified, including 47 prototypes and 71 metabolites, among which 61 unknown compounds were reasonably characterized. The typical compound identified by this strategy was successfully validated using a triterpene standard. This strategy can improve the annotation confidence of in vivo metabolites of TCM and facilitate further pharmacological research.

Metabolic profile of curcumin self‐emulsifying drug delivery system in rats determined by ultra‐high performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry

Curcumin (Cur) is a natural anticancer pigment, but its poor absorption and extensive metabolism limit its clinical applications. In this study, an ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry method was employed to investigate the metabolic profiles of a Cur self-emulsifying drug delivery system (C-SEDDS) in rat plasma, urine, bile and feces after oral administration at 100 mg/kg. Protein precipitation, solid-phase and ultrasonic extractions were used to prepare different biosamples. A total of 34 metabolites were identified using available reference standards, or tentatively identified based on the mass spectrometric fragmentation patterns and the chromatographic elution order. Nine metabolites of Cur were found for the first time in vivo. Glucuronidation, sulfation, reduction, dehydroxylation, demethylation, demethoxylation and methylation were its possible metabolic reactions. Moreover, the differences were compared in terms of plasma metabolites found in C-SEDDS-treated, Cur suspension-treated and rats treated with a commercial curcuminoid phospholipid complex administered at the same oral dose. Dihydrocurcumin (DHC), DHC glucuronide and methylated DHC were found only in the metabolic profile of C-SEDDS-treated rat plasma, suggesting that different drug delivery systems may cause a change in Cur metabolic pathways. This study provides a sensitive and rapid method for the identification of Cur metabolites in biosamples.

Comparative metabolism of schaftoside in healthy and calcium oxalate kidney stone rats by UHPLC-Q-TOF-MS/MS method

Schaftoside is a flavone-C-glycoside isolated from Herba Desmodii Styracifolii with valuable anti-kidney stones efficacies. In this study, a six-step strategy was first developed to detect and identify the metabolites in plasma, urine, bile, feces and rat intestinal bacteria samples of healthy and model rats administrated with schaftoside using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). The number and the relative peak area of metabolites in healthy rats and model rats were compared, and it was noticed that metabolites in bio-samples of healthy and model rats both had obvious differences. A total of 28 metabolites of schaftoside in healthy rats and 30 metabolites in model rats were initially indentified. The relative peak area of the parent drug and every metabolite in model rat plasma samples were larger than those in healthy rat plasma. Those metabolites with high blood concentrations might be beneficial for the treatment of calcium oxalate stones in the kidney. The results are valuable and important for understanding the metabolic process of schaftoside in clinical application, and especially the metabolism study in calcium oxalate kidney stone model rats could provide a beneficial reference for the further search of effective substances associated with the treatment of kidney stones.

Selective extraction and determination of aromatic amine metabolites in urine samples by using magnetic covalent framework nanocomposites and HPLC-MS/MS.

Purification and selective enrichment of ultra-low level metabolites in bio-samples is very important for HPLC-MS/MS analysis. A magnetic covalent organic framework (i.e. COFs) (TpPa-1) (i.e. 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde-p-phenylenediamine) material was synthesized and used for a magnetic solid phase extraction (MSPE) method in aromatic amine (AA) metabolites (i.e. 1-naphthylamine, 2-naphthylamine, 3-aminobiphenyl and 4-aminobiphenyl) in urine, and then HPLC-MS/MS was employed for analysis. The morphology, pore structure, surface area, chemical composition, magnetic properties, and thermal stability of the synthesized magnetic COFs were characterized with several analysis techniques, such as nitrogen adsorption–desorption isotherms, HRTEM, FTIR, XRD, and so on. MSPE conditions were optimized and analytical performance of the developed method was characterized. The limit of detection (LOD) and limit of quantification (LOQ) of four AAs ranged from 0.01 to 0.07 ng mL−1 and 0.04 to 0.22 ng mL−1, respectively. The recoveries of 1-NA, 2-NA, 3-ABP, 4-ABP were in the range of 81.9–105%, 87.8–102%, 101–120%, 88.3–117%, respectively. Good intra-day and inter-day precision were obtained with RSD of less than 5.8% and 9.9%, respectively. Furthermore, the synthesized magnetic COFs absorbent could be recycled in MSPE at least 5 times. Analytical results of AA metabolites in real urine samples with the developed method showed significant difference (p < 0.01) between smokers and nonsmokers. Thus, urinary AA metabolites could be exposure biomarkers for cigarette smoke.

Enhanced identification of the in vivo metabolites of Ecliptae Herba in rat plasma by integrating untargeted data-dependent MS2 and predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion scan

Detection and identification of the in vivo metabolites of traditional Chinese medicine by untargeted profiling strategies are often confronted with severe interference from complex endogenous substances. Here we developed an integral approach, by combining untargeted data-dependent MS2 (dd-MS2) of Q-Orbitrap mass spectrometry and predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion scan (pMRM-IDA-EPI) of triple quadrupole-linear ion trap (QTRAP) mass spectrometry, aiming to detect and identify more extensive metabolites in bio-samples. Ecliptae Herba (EH) is a widely consumed medicinal herb with the effects of nourishing liver/kidney, but its metabolites in vivo have not been fully elucidated. Firstly, after UHPLC separation on an HSS T3 column, chemical fingerprinting of 70% ethanolic extract of EH was performed by untargeted dd-MS2 in negative ion mode. We could characterize 41 compounds from EH, and 24 were detectable in the plasma of rats (prototypes) after oral administration of EH extract (1 g/kg). Secondly, using echinocystic acid (triterpene), wedelolactone (coumarin), and apigenin (flavonoid) as the different parent templates, an MRM list containing 150 predicted ion-pairs was established to enhance MS2 scan by pMRM-IDA-EPI, which enabled the primary identification of up to 200 metabolites. The biotransformations mainly involve oxidation, hydrogenation, methylation, glucuronidation, sulfonation etc. Thirdly, the rat plasma samples obtained after oral administration of three pure compounds (echinocystic acid, wedelolactone and apigenin) were analyzed to verify the reliability of metabolites identification, and 11, 4, and 10 metabolites were found individually. This is the first comprehensive research on the metabolism of EH in vivo.

Development of a Data-Independent Targeted Metabolomics Method for Relative Quantification Using Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Quantitative metabolomics approaches can significantly improve the repeatability and reliability of metabolomics investigations but face critical technical challenges, owing to the vast number of unknown endogenous metabolites and the lack of authentic standards. The present study contributes to the development of a novel method known as "data-independent targeted quantitative metabolomics" (DITQM), which was used to investigate the label-free quantitative metabolomics of multiple known and unknown metabolites in biofluid samples. This approach initially involved the acquisition of MS/MS data for all metabolites in biosamples using a sequentially stepped targeted MS/MS (sst-MS/MS) method, in which multiple product ion scans were performed by selecting all ions in the targeted mass ranges as the precursor ions. Subsequently, scheduled multiple reaction monitoring (MRM) by LC-MS/MS of the metabolome was established for 1658 characteristic ion pairs of 1324 metabolites. For sensitive and accurate quantification of these metabolites, mixed calibration curves were generated using sequentially diluted standard reference plasma samples using established MRM methods. Relative concentrations of all metabolites in each sample were calculated without using individual authentic standards. To evaluate the reliability and applicability of this new method, the performance of DITQM was validated by comparison to absolute quantification of 12 acylcarnitines using authentic standards and traditional metabolomics analysis for lung cancer. The results proved that the DITQM protocol is more reliable and can significantly improve clustering effects and repeatability in biomarker discovery. In this study, we established a novel methodology to standardize and quantify large-scale metabolome, providing a new choice for metabolomics research and its clinical applications.

A pre-classification strategy based on UPLC-Triple-TOF/MS for metabolic screening and identification of Radix glehniae in rats.

Traditional Chinese Medicines (TCMs) have gained increasing popularity in modern society. However, the profiles of TCMs in vivo are still unclear owing to their complexity and lowlevel in vivo. In this study, UPLC-Triple-TOF techniques were employed for data acquiring, and a novel pre-classification strategy was developed to rapidly and systematically screen and identify the absorbed constituents and metabolites of TCMs in vivo using Radix glehniae as the research object. In this strategy, pre-classification for absorbed constituents was first performed according to the similarity of their structures. Then representative constituents were elected from every class and analyzed separately to screen non-target absorbed constituents and metabolites in biosamples. This pre-classification strategy is basing on target (known) constituents to screen non-target (unknown) constituents from the massive data acquired by mass spectrometry. Finally, the screened candidate compounds were interpreted and identified based ona predicted metabolic pathway, well - studied fragmentation rules,a predicted metabolic pathway, polarity and retention time of the compounds, and some related literature. With this method, a total of 111 absorbed constituents and metabolites of Radix glehniae in rats' urine, plasma, and bile samples were screened and identified or tentatively characterized successfully. This strategy provides an idea for the screening and identification of the metabolites of other TCMs.

Polyamine Metabolites Profiling for Characterization of Lung and Liver Cancer Using an LC-Tandem MS Method with Multiple Statistical Data Mining Strategies: Discovering Potential Cancer Biomarkers in Human Plasma and Urine

Polyamines, one of the most important kind of biomarkers in cancer research, were investigated in order to characterize different cancer types. An integrative approach which combined ultra-high performance liquid chromatography—tandem mass spectrometry detection and multiple statistical data processing strategies including outlier elimination, binary logistic regression analysis and cluster analysis had been developed to discover the characteristic biomarkers of lung and liver cancer. The concentrations of 14 polyamine metabolites in biosamples from lung (n = 50) and liver cancer patients (n = 50) were detected by a validated UHPLC-MS/MS method. Then the concentrations were converted into independent variables to characterize patients of lung and liver cancer by binary logic regression analysis. Significant independent variables were regarded as the potential biomarkers. Cluster analysis was engaged for further verifying. As a result, two values was discovered to identify lung and liver cancer, which were the product of the plasma concentration of putrescine and spermidine; and the ratio of the urine concentration of S-adenosyl-l-methionine and N-acetylspermidine. Results indicated that the established advanced method could be successfully applied to characterize lung and liver cancer, and may also enable a new way of discovering cancer biomarkers and characterizing other types of cancer.

Metabolic fingerprinting to understand therapeutic effects and mechanisms of silybin on acute liver damage in rat.

Background:Metabolic fingerprinting is a rapid and noninvasive analysis, representing a powerful approach for the characterization of phenotypes and the distinction of specific metabolic states due to environmental alterations. It has become a valuable analytical approach for the characterization of phenotypes and is the rapidly evolving field of the comprehensive measurement of ideally all endogenous metabolites in bio-samples. Silybin has displayed bright prospects in the prevention and therapy of liver injury, and we had conducted a preliminary exploration on the molecular mechanism of the hepatoprotective effects of silybin. Because the knowledge on the metabolic responses of an acute liver damage rat to the silybin is still scarce, metabolic fingerprinting can provide relevant information on the intrinsic metabolic adjustments.Materials and Methods:Here, the physiological and metabolic changes in the acute liver damage rat were investigated by performing a metabolic analysis. The phenotypic response was assessed by liquid chromatography/mass spectrometry (LC/MS) combined with pattern recognition approaches such as principal components analysis and partial least squares projection to supervised latent structures and discriminant analysis. Multivariate analysis of the data showed trends in scores plots that were related to the concentration of the silybin.Results:Results indicate 10 ions (7 upregulated and 3 downregulated) as differentiating metabolites. Key observations include perturbations of metabolic pathways linked to glutathione metabolism, tryptophan metabolism, cysteine and methionine metabolism, etc., Overall, this investigation illustrates the power of the LC/MS combined with the pattern recognition methods that can engender new insights into silybin affecting on metabolism pathways of an acute liver damage rat.Conclusion:The present study demonstrates that the combination of metabolic fingerprinting with appropriate chemometric analysis is a valuable approach for studying cellular responses to silybin drug and can provide additional insight into the mechanisms.

Current position of high-resolution MS for drug quantification in clinical & forensic toxicology.

This paper reviews high-resolution MS approaches published from January 2011 until March 2014 for the quantification of drugs (of abuse) and/or their metabolites in biosamples using LC-MS with time-of-flight or Orbitrap™ mass analyzers. Corresponding approaches are discussed including sample preparation and mass spectral settings. The advantages and limitations of high-resolution MS for drug quantification, as well as the demand for a certain resolution or a specific mass accuracy are also explored.

Metabolite Profiling and Pharmacokinetics of Herbal Compounds Following Oral Administration of a Cardiovascular Multi-herb Medicine (Qishen Yiqi Pills) in Rats

Qishen yiqi pills (QY pills) are a type of standardized cardiovascular herbal medicine, which contain four component herbs, i.e., Astragalus membranaceus (Huangqi), Salvia miltiorrhiza (Danshen), Panax notoginseng (Sanqi), and Dalbergia odorifera (Jiangxiang). After oral administration of QY pills, the in vivo exposure types of each component herb in rats were first uncovered and identified according to a target-directed strategy based on hyphenated chromatography techniques. The dominated metabolites in urine, blood and bile were originated from flavonoids of Huangqi and monomer phenolic acids of Danshen; no metabolites but parent drugs of Sanqi ginsenosides, namely ginsenosides Rb1, Rd, Re and Rg1, notoginsenoside R1 and gypenoside XVII, were detected in rat urine and blood, and the 20(S)-protopanaxatriol type ginsenosides (NR1, GRe, GRg1) could also be excreted to bile; the high liposolubility of volatile oils from Jiangxiang restricted them to small intestine, liver and adipose tissues. The identification of metabolites in bio-samples was achieved by exact mass measurement and detailed fragmentation pathway analyses. In specific conditions, not only the types of phase II metabolism but also their conjugation positions could be determined by our established cleavage pathways, which lead to discriminate the phase II metabolites of protocatechualdehyde for the first time. Based on the metabolite study in rats, the 4 main compounds (tanshinol, astragaloside IV, GRb1 and GRg1) in QY pills were selected as pharmacokinetic markers. The PK results showed that their maximal concentrations in blood were obtained within one hour, much shorter than the reported values in single herbs. The rat exposure was proximately linear under the studied dosages from 1 to 6 g/kg.

The Use of Thin-Layer Chromatography in Experimental Xenobiology

Abstract Studies of the metabolism and disposition of most drugs or other xenobiotics share one common feature: the need for an analytical method to measure the xenobiotic and/or its biotransformation products in biosamples. Although chromatographic methods such as GC and HPLC are important and preferred by some laboratories, initial investigations of new xenobiotic entities are often conducted using radiolabeled compounds, and, in such instances, TLC assay methods are frequently selected. TLC is utilized in xenobiotic metabolism and disposition studies to evaluate the radiochemical purity of labeled xenobiotics, the extent of metabolism, the profile of metabolites in blood and excreta, the concentrations of the xenobiotic and specific metabolites in blood and other biosamples, the stability of the xenobiotic and its metabolites in biosamples, and the relative behavior of metabolites and reference compounds. Preparative TLC is frequently used to isolate specific metabolites. Thin-layer radiochromatography (TLRC) has been utilized to study the pharma-cokinetics of drugs and other xenobiotics in man and in animals. Especially where radiolabeled compounds are used, TLC provides an attractive chromatographic alternative to GC and HPLC in research in experimental xenobiology.