Interaction Liquid Chromatography Tandem Mass Research Articles

Development and validation of an HILIC/MS/MS method for determination of nusinersen in rabbit plasma

A hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC/MS/MS) method was developed and validated for the quantitative analysis of the fully phosphorothioate modified oligonucleotide nusinersen. HILIC/MS/MS method is more robust and compatible with mass spectrometry than ion pair reversed-phase liquid chromatography-tandem mass spectrometry (IP-RP-LC/MS/MS). Various types and concentrations of additives and different pH of mobile phase affected the mass spectrometry response, chromatographic peak shape and retention of nusinersen. The optimized extraction method of nusinersen employs hydrophilic-lipophilic balance solid phase extraction, with a recovery of up to 80%. Chromatographic quantification was performed using a gradient system on an amide column and the mobile phase consisted of ammonium acetate, acetonitrile and water in a certain proportion. The fully phosphorothioate modified nusinersen can obtain a high mass spectrometry response by providing greater peak symmetry and high ionization efficiency in a high-pH mobile phase. Moreover, the significant carry over interference was observed at the pH 6.3 of the mobile phase. Adjusting the pH value up to 10, and the carry over interference disappeared. The lower limit of quantitation of this developed HILIC/MS/MS assay was 30.0 ng/mL and the method was systematic methodology validated. This HILIC/MS/MS method provides an attractive and robust alternative for the quantitative analysis of nusinersen and was applied in the pharmacokinetic study of nusinersen in rabbits.

Highly sensitive and accurate measurement of underivatized phosphoenolpyruvate in plasma and serum via EDTA-facilitated hydrophilic interaction liquid chromatography–tandem mass spectrometry

Phosphoenolpyruvate (PEP) is an essential intermediate metabolite that is involved in various vital biochemical reactions. However, achieving the direct and accurate quantification of PEP in plasma or serum poses a significant challenge owing to its strong polarity and metal affinity. In this study, a sensitive method for the direct determination of PEP in plasma and serum based on ethylenediaminetetraacetic acid (EDTA)-facilitated hydrophilic interaction liquid chromatography–tandem mass spectrometry was developed. Superior chromatographic retention and peak shapes were achieved using a zwitterionic stationary-phase HILIC column with a metal-inert inner surface. Efficient dechelation of PEP–metal complexes in serum/plasma samples was achieved through the introduction of EDTA, resulting in a significant enhancement of the PEP signal. A PEP isotopically labelled standard was employed as a surrogate analyte for the determination of endogenous PEP, and validation assessments proved the sensitivity, selectivity, and reproducibility of this method. The method was applied to the comparative quantification of PEP in plasma and serum samples from mice and rats, as well as in HepG2 cells, HEK293T cells, and erythrocytes; the results confirmed its applicability in PEP-related biomedical research. The developed method can quantify PEP in diverse biological matrices, providing a feasible opportunity to investigate the role of PEP in relevant biomedical research.

Hepatic Topology of Glycosphingolipids in Schistosoma mansoni-Infected Hamsters.

Schistosomiasis is a neglected tropical disease caused by worm parasites of the genus Schistosoma. Upon infection, parasite eggs can lodge inside of host organs like the liver. This leads to granuloma formation, which is the main cause of the pathology of schistosomiasis. To better understand the different levels of host-pathogen interaction and pathology, our study focused on the characterization of glycosphingolipids (GSLs). For this purpose, GSLs in livers of infected and noninfected hamsters were studied by combining high-spatial-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) with nanoscale hydrophilic interaction liquid chromatography tandem mass spectrometry (nano-HILIC MS/MS). Nano-HILIC MS/MS revealed 60 GSL species with a distinct saccharide and ceramide composition. AP-SMALDI MSI measurements were conducted in positive- and negative-ion mode for the visualization of neutral and acidic GSLs. Based on nano-HILIC MS/MS results, we discovered no downregulated but 50 significantly upregulated GSLs in liver samples of infected hamsters. AP-SMALDI MSI showed that 44 of these GSL species were associated with the granulomas in the liver tissue. Our findings suggest an important role of GSLs during granuloma formation.

Circulating choline and phosphocholine measurement by a hydrophilic interaction liquid chromatography–tandem mass spectrometry

BackgroundGiven the growing interest in studying the role of choline and phosphocholine in the development and progression of tumor pathology, in this study we describe the development and validation of a fast and robust method for the simultaneous analysis of choline and phosphocholine in human plasma. MethodsCholine and phosphocholine quantification in human plasma was obtained using a hydrophilic interaction liquid chromatography–tandem mass spectrometry technique. Assay performance parameters were evaluated using EMA guidelines. ResultsCalibration curve ranged from 0.60 to 38.40 μmol/L (R2 = 0.999) and 0.08–5.43 μmol/L (R2 = 0.998) for choline and phosphocholine, respectively. The Limit Of Detection of the method was 0.06 μmol/L for choline and 0.04 μmol/L for phosphocholine. The coefficient of variation range for intra-assay precision is 2.2–4.1 % (choline) and 3.2–15 % (phosphocholine), and the inter-assay precision range is < 1–6.5 % (choline) and 6.2–20 % (phosphocholine). The accuracy of the method was below the ±20 % benchmarks at all the metabolites concentration levels. In-house plasma pool of apparently healthy adults was tested, and a mean concentration of 15.97 μmol/L for Choline and 0.34 μmol/L for Phosphocholine was quantified. ConclusionsThe developed method shows good reliability in quantifying Choline and Phosphocholine in human plasma for clinical purposes.

O-288 Implantation, ultrastructure and metabolic profiling of embryos from PGT-M and PGT-A Cycles

Abstract Study question Can metabolic profiling predict embryos at risk of single gene defects and chromosomal abnormalities and how are these reflected in their ultrustructure? Summary answer Different metabolic profiles are observed between embryos with chromosomal abnormalities and monogenic disorders and normal embryos, linked to altered mitochondrial and other organelles’ structure/function What is known already Down’s syndrome embryos and Monosomy 21 embryos have previously been shown to have differential expression of metabolites compared to normal embryos, but limited studies, have investigated in detail the metabolic profiling of embryos with other abnormalities or those with monogenic disorders in comparison to chromosomally normal embryos or their reflection in their ultrustructure. Study design, size, duration 520 embryos were biopsied on day5 for PGT-A (n = 178-47 cycles) or on day3 for PGT-A (n = 178-33 cycles) and PGT-M (n = 164-24 cycles). Following transfer of normal embryos, spare embryos, rejected for transfer following day5 or day3 biopsy were processed for TEM (n = 60 day3 biopsied, n = 60 day5 biopsied/vitrified), 60 unbiopsied/vitrified embryos from egg donation cycles used as control. Culture media were collected from the embryos and analysed by hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS). Participants/materials, setting, methods PGT-A and PGT-M were performed in 2 IVF Units. Metabolic profiling was conducted in a Forensic Toxicology Laboratory by HILIC-MS/MS (100 metabolites). Ultrastructure analysis by TEM was carried out in an Academic Hospital and Histology/Embryology Laboratory following embryo fixation in 3% glutaraldehyde, 1% osmium tetroxide, washes in PBS and staining with 1% aqueous uranyl acetate. Main results and the role of chance The implantation rates (+ve hCG/ET) for the PGT-M cycles was 68.2% and the for the PGT-A (blastocyst and cleavage biopsy cycles) 64.5% and 65% respectively. Characteristic patient specific metabolic profiles after screening for &amp;gt;100 primary metabolites were observed which differed between normal embryos that had resulted in a viable pregnancy and aneuploid and chaotic embryos although it was more difficult to find clear patterns in embryos with monogenic disorders. Logistic regression analysis revealed a number of metabolites with high predictive value which in combination with embryo score could serve in the future as non-invasive markers for the detection of chromosomal abnormalities before embryo transfer. TEM analysis revealed differences in the quality of cells and organelle activity which were reflected in the embryo metabolic profiles. Abnormal but well developed hatching blastocysts had mainly cells with good mitochondrial morphology/ activity, nice Golgi apparatus and well developed rough and smooth endoplasmic reticulum but depending on the aneuploidy or gene mutation involved, inner cell mass cells with limited organelles and occasionally lipofuscin droplets in the trophectoderm were evident. Chaotic poor quality embryos showed a lower number of mitochondria, often with no cisternae, increased number of vacuoles, and frequently problems in junctions between cells. Limitations, reasons for caution Although metabolic profiles were compared between normal and abnormal embryos, all the normal embryos were transferred to the uterus or remain vitrified for clinical purposes. Therefore the ultrastructure analysis is based only on biopsied abnormal embryos and as control unbiopsied embryos from egg-donation cycles with high chance of being normal. Wider implications of the findings This study shows high implantation rates after PGT—M and PGT-A and identified distinct differences in the metabolic profiles of normal and abnormal embryos providing unique metabolites which in the future could serve as non-invasive biomarkers for the detection of abnormalities before embryo transfer. Trial registration number Not applicable

The Maillard reaction end product Nε-carboxymethyllysine is metabolized in humans and the urinary levels of the microbial metabolites are associated with individual diet.

During food processing most of the thermally-driven chemical reactions start off on the side chain amino group of lysine generating structurally modified compounds with specific metabolic routes. Upon human digestion, dietary Nε-carboxymethyllysine (CML) may enter the colon and undergo gut microbial metabolism. However, little is known about the in vivo metabolic fate of dietary CML and its relationship with the habitual diet. We explored by hydrophilic interaction liquid chromatography tandem mass spectrometry the metabolites of CML in urine samples collected from 46 healthy subjects and studied the associations with diet. Mean concentration of N-carboxymethylcadaverine (CM-CAD), N-carboxymethylaminopentanoic acid (CM-APA), N-carboxymethylaminopentanol (CM-APO), and the N-carboxymethyl-Δ1-piperideinium ion were 0.49 nmol mg-1 creatinine, 1.45 nmol mg-1 creatinine, 4.43 nmol mg-1 creatinine and 4.79 nmol mg-1 creatinine, respectively. The urinary concentration of CML, its metabolites and lysine were positively correlated. Dietary intake of meat products negatively correlated with urinary excretion of CML and CM-APA; conversely dietary plant-to-animal proteins ratio positively correlated with urinary CML and its metabolites. The identification and quantification of CML metabolites in urine and the associations with diet corroborate the hypothesis that CML, an advanced glycation end-product, can undergo further biochemical transformations in vivo. The gut microbiome may have a major role in human metabolism of dietary CML.

Analysis of degradation products of Novichok agents in human urine by hydrophilic interaction liquid chromatography–tandem mass spectrometry

PurposeThe detection of hydrolysis products of Novichok agents in biological samples from victims is important for confirming exposure to these agents. However, Novichok agents are new class of nerve agent and there have been only few reports on analyses of Novichok agent degradation products. Here, we developed hydrophilic interaction liquid chromatography (HILIC)–tandem mass spectrometry (MS/MS) methods to detect Novichok agent degradation products in human urine with simple pretreatment and high sensitivity.MethodsA Poroshell 120 HILIC-Z column was used to analyze six Novichok agent degradation products. For urine samples, we used a simple pretreatment method, which consisted of deproteinization with acetonitrile and microfiltration. We calculated the pKa values of the OH groups, the log P values, and the molecular weights to investigate the difference in chromatographic behaviors of the Novichok agent degradation products and the degradation products of conventional nerve agents.ResultsSix Novichok agent degradation products, including N-(bis-(diethylamino)methylidene)-methylphosphonamidic acid (MPGA), which could not be detected by our previous method, could be analyzed with sufficient peak shape and mutual separation. The detection limits of six Novichok agent degradation products were sufficiently low (1–50 ng/mL) and the calibration curves showed sufficient linearity. The physicochemical parameters of Novichok agent degradation products were different from those of conventional nerve agent degradation products, and this explains the difference in chromatographic behaviors.ConclusionSix Novichok agent degradation products were successfully analyzed by HILIC–MS/MS. Due to the absence of a derivatization step, throughput performance was higher than our previous derivatization-liquid chromatography–MS/MS method.

Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry of 5-Aminolevulinic Acid for Low Concentration and Small Sample Volumes

Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry of 5-Aminolevulinic Acid for Low Concentration and Small Sample Volumes

Simultaneous Determination of Methylated Nucleosides by HILIC-MS/MS Revealed Their Alterations in Urine from Breast Cancer Patients.

RNA methylation plays a vital role in the pathogenesis of a variety of diseases including cancer, and aberrant levels of modified nucleosides in RNA were revealed to be related to cancer. Urine is a favored source for biomarker discovery due to the non-invasion to patients. Herein, we developed a sensitive hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC–MS/MS) method combined with stable isotope dilution for accurate quantification of methylated nucleosides in human urine. With this method, we successfully quantified ten methylated nucleosides in urine samples collected from healthy controls and breast cancer patients. We found N6-methyladenosine (m6A), 2′-O-methyladenosine (Am), N1-methyladenosine (m1A), N6,2′-O-dimethyladenosine (m6Am), N1-methylguanosine (m1G), 2′-O-methylguanosine (Gm), 5-methylcytidine (m5C) and 2′-O-methylcytidine (Cm) were all decreased in early-stage breast cancer patients, and a nomogram prediction model was constructed. Locally advanced breast cancer patients exhibited elevated levels of urinary 2′-O-methylated nucleosides in comparison to early-stage breast cancer patients. Together, we developed a robust method for the simultaneous determination of methylated nucleosides in human urine, and the results revealed an association between the contents of urinary methylated nucleosides and the occurrence of breast cancer, which may stimulate future studies about the regulatory roles of these methylated nucleosides in the initiation and progression of breast cancer.

O-174 The association of chromosomal abnormalities with embryo ultrastructure, spindle anomalies and metabolic profiling

Abstract Study question Can metabolic profiling predict embryos at risk of chromosomal abnormalities and how are these reflected in the ultrustructure and cytoskeleton? Summary answer Different metabolic profiles are observed between normal and aneuploid/chaotic embryos which are linked to altered mitochondrial and other organelles’ structure/function and spindle and nuclear abnormalities. What is known already One of the greatest challenges in IVF is the selection of the best ‘fit’ embryo for implantation in a non-invasive way. Down’s syndrome embryos and Monosomy 21 embryos have previously been shown to have differential expression of metabolites compared to normal embryos, but limited studies, have investigated in detail the metabolic profiling of embryos with other abnormalities in comparison to chromosomally normal embryos or their reflection in the ultrustructure and the cytoskeleton. Study design, size, duration Culture media collected on day 3 from 200 embryos which underwent PGT-A, were analysed by hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS). The chromosomally normal embryos were transferred to the uterus (1–2 embryos/ET) or still remain vitrified for clinical use and 120 of the embryos that were diagnosed with chromosomal abnormalities were either processed for spindle/chromosome configurations analysis (n = 60) by confocal laser scanning microscopy(CLSM) or for ultrastructural analysis (n = 60) by Transmission Electron Microscopy(TEM). Participants/materials, setting, methods Metabolic profiling was conducted in a Forensic Toxicology Laboratory by HILIC-MS/MS (100 metabolites). Spindle Chromosome Configuration analysis was conducted in an academic hospital after methanol fixation and immunostaining with α-tubulin, γ-tubulin, acetylated-tubulin antibodies and DAPI or/ PI to visualise DNA. Ultrastructure analysis by TEM was carried out in a Histology/Embryology Laboratory following embryo fixation in 3% glutaraldehyde, 1% osmium tetroxide, washes in PBS and staining with 1% aqueous uranyl acetate. Main results and the role of chance This study provides screening for &amp;gt;100 primary metabolites using HILIC-MS/MS in a single run of 40 minutes. Characteristic patient specific metabolic profiles were observed which differed between normal embryos that had resulted in a viable pregnancy and aneuploid and chaotic embryos. Logistic regression analysis revealed a number of metabolites that had a high predictive value including Isoleucine, lysine and glucose and models were created in combination with embryo score which in the future could serve as non-invasive markers for the detection of chromosomal abnormalities before embryo transfer. TEM analysis revealed differences in the quality of cells and organelle activity which were reflected in the embryo metabolic profiles. Chaotic poor quality embryos showed a lower number of mitochondria, often with no cisternae, increased number of vacuoles, and frequently problems in junctions between cells. Aneuploid but well developed hatching blastocysts had mainly cells with good mitochondrial morphology/ activity, nice Golgi apparatus and well developed rough and smooth endoplasmic reticulum but depending on the aneuploidy involved, inner cell mass cells with limited organelles and occasionally lipofuscin droplets in the trophectoderm were evident. Nuclear and chromosomal abnormalities were interrelated through abnormalities in cytokinesis and spindle formation and reflected in the embryo metabolic profiles. Limitations, reasons for caution Although metabolic profiles were compared between normal and chromosomally abnormal embryos identified by PGT-A, all the normal embryos were transferred to the uterus or remain vitrified for clinical purposes and therefore the ultrastructure analysis and the spindle chromosome configuration analysis are based only on chromosomally abnormal embryos. Wider implications of the findings This study identified distinct differences in the metabolic profiles of normal and chromosomally abnormal embryos and provides unique metabolites which in the future could serve as non-invasive biomarkers for the detection of chromosomal abnormalities before embryo transfer. Trial registration number not applicable

Design and validation of an LC-MS/MS method for simultaneous quantification of asymmetric dimethylguanidino valeric acid, asymmetric dimethylarginine and symmetric dimethylarginine in human plasma

Asymmetric dimethylguanidino valeric acid (ADGV), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are three arginine metabolites which have utility in the assessment of cardiovascular disease, renal disease and non-alcoholic fatty liver disease (NAFLD). Translation of these research metabolomic markers into routine clinical use requires the development of robust assays with appropriately assessed preanalytical variables and traceable clinical reference intervals. A hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ADGV, ADMA and SDMA was developed. Sample stability and collection conditions were scrutinised to determine any preanalytical factors that could affect quantification under routine laboratory conditions. Patient samples from 120 males and 120 females were used to derive preliminary reference intervals. All three analytes were quantifiable in human plasma using unique MS/MS transitions. The analytes were stable for up to a week once separated from red cells, though reduced stability was observed upon extraction of the analytes from plasma. The assay was linear for concentration of ADGV between 1.6nmol/L and 200nmol/L and for ADMA and SDMA between 0.1μmol/L and 4.0μmol/L. The accuracy for all analytes was 97-103% and interday and intraday imprecisions (coefficients of variation) were less than 10%. ADGV concentrations were noted to be lower in the female reference population when compared to males. The analytical method shows excellent performance and is sufficiently robust to be used in the clinical investigation of cardiovascular disease and NAFLD.

Development of a Simple, Underivatized Method for Rapid Determination of Free Amino Acids in Honey Using Dilute-and-Shoot Strategy and Liquid Chromatography-Tandem Mass Spectrometry.

A simple, fast and reliable analytical method was developed for 20 free amino acids (FAAs) determination in honey samples through a dilute-and-shoot strategy and hydrophilic interaction liquid chromatography tandem mass spectrometry. Compared with previous reports, direct dilution by water has significantly reduced the matrix effect and facilitated full extraction of FAAs. Further, a 5 min determination method was established with an acetonitrile–water mobile phase system with 0.1% formic acid addition. The established method was validated and demonstrated several advantages including short detection time, wide linear range over 3–4 orders of magnitude, high sensitivity down to 0.1 ng/mL and negligible matrix effect. Twenty FAAs were determined in 10 honey samples from different botanical origins by this method, and 19 FAAs were found. This general applicable method was also promising for fast determination of FAAs in other practical samples.

Magnetic Amino-Modified Multiwalled Carbon Nanotube (MWCNT) Based Magnetic Dispersive Solid-Phase Extraction (m-dSPE) for the Determination of Paralytic Shellfish Toxins in Bivalve Mollusks with Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry (HILIC-MS/MS)

A sensitive and efficient magnetic amino modified multi-walled carbon nanotubes (m-MWCNT-NH2)-based magnetic dispersive solid-phase extraction (m-dSPE) coupled with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) strategy is reported for the simultaneous determination of 13 paralytic shellfish toxins (PSTs) in bivalve mollusks. The samples were extracted twice with 1% acetic acid and purified using optimized m-dSPE procedures with Fe3O4 coated MWCNT-NH2 composites as magnetic adsorbents. The adsorbed PSTs on the m-MWCNT-NH2 adsorbents were separated using a Ni-coated neodymium magnet and subsequently eluted with 2 mL water–acetonitrile–acetic acid (80:20:1, v/v/v) and analyzed by HILIC-MS/MS. The correlation coefficients (r) of the targeted toxins obtained in matrix-matched external standard curves were from 0.997 to 0.999. The PSTs were acquired and quantified in the multiple reaction-monitoring mode and the limits of detection and quantitation in bivalve mollusks were 1.10 to 4.51 μg/kg and 3.67 to 13.5 μg/kg, respectively. Spiked recoveries at three concentrations in negative samples were from 73.4% to 92.5% with precision less than 10.6%. The method validation was in accordance with 2002/657/EC guidelines and method application in commercial bivalve mollusk samples. The developed method was rapid, convenient and economical for the determination of the targeted toxins.

Determination of Fosetyl-Aluminum in Wheat Flour with Extract-Dilute-Shoot Procedure and Hydrophilic Interaction Liquid Chromatography Tandem Mass Spectrometry

Fosetyl-aluminum is a widely used ionic fungicide. This pesticide is not amenable to the common multi-residue sample preparation methods. Herein, this paper describes a novel method for the simple and sensitive determination of fosetyl-aluminum residue in wheat flour. The sample preparation method involved extraction with water under ultrasonication and subsequent dilution with six-fold acetonitrile. The fosetyl-aluminum concentration was determined by hydrophilic interaction liquid chromatography tandem mass spectrometry. The limit of detection and quantification were only 5 and 10 ng/g, respectively, which meet the requirement of the current European legislation. Matrix-matched linearity (r2 = 0.9999) was established in the range of 10–2000 ng/g. Satisfactory recoveries were achieved in the range of 95.6% to 105.2% for three levels of spiked samples (10, 50, and 100 ng/g). Finally, the method was applied to analyzing 75 wheat flour samples produced in four provinces in China. Two samples were positive with concentrations over the limit of detection. This is the first method focusing on fosetyl-aluminum determination in wheat flour with an extract-dilute-shoot strategy and is very promising for the routine quality control of fosetyl-aluminum in similar cereal matrices.

Mass Spectrometry-Based Targeted Serum Monomethylated Ribonucleosides Profiling for Early Detection of Breast Cancer.

RNA methylation plays a significant regulatory role in various of physiological activities and it has gradually become a hotspot of epigenetics in the past decade. 2′-O-methyladenosine (Am), 2′-O-methylguanosine (Gm), 2′-O-methylcytidine (Cm), 2′-O-methyluridine (Um), N 6-methyladenosine (m6A), N 1-methylguanosine (m1G), 5-methylcytidine (m5C), and 5-methyluridine (m5U) are representative 2′-O-methylation and base-methylation modified epigenetic marks of RNA. Abnormal levels of these ribonucleosides were found to be related to various diseases including cancer. Serum is an important source of biofluid for the discovery of biomarkers, and novel tumor biomarkers can be explored by measuring these ribonucleoside modifications in human serum. Herein, we developed and applied a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method to determine the content of monomethylated ribonucleosides in human serum. The developed method enabled sensitive and accurate determination of these monomethylated ribonucleosides. By applying this robust method, we demonstrated the presence of Gm and Um in human serum for the first time, and we successfully quantified m6A, Gm, m1G, Cm, Um and m5U in serum samples collected from 61 patients with breast cancer and 69 healthy controls. We discovered that the levels of Gm, m1G, Cm, Um and m5U in serum were all significantly decreased in breast cancer patients whereas m6A was increased. We performed receiver operating characteristic (ROC) curve analysis, and obtained highest area under curve (AUC) value when combining these six monomethylated ribonucleosides together. These results suggest that m6A, Gm, m1G, Cm, Um and m5U might have great potential to be novel biomarkers for detection of breast cancer in the early stage. In addition, this study may stimulate future investigations about the regulatory roles of monomethylated ribonucleosides on the initiation and development of breast cancer.

Comprehensive characterization of the chemical constituents in Platycodon grandiflorum by an integrated liquid chromatography-mass spectrometry strategy

Platycodon grandiflorum (PG), as a well-known medicine food homology species, possess various pharmacological effects and health benefits. Aiming to facilitate in-depth and global characterization of the chemical compositions of PG, a profiling method based on ultra-high performance liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry (UPLC/IM-QTOF-MS) was conducted. Consequently, as many as 187 compounds were plausibly or unambiguously identified. Most importantly, phospholipids (PLs) were first observed and identified in PG. Due to their widely confirmed bioactivities, an analysis scheme was developed by hydrophilic interaction liquid chromatography and electrospray ionization tandem mass spectrometry combined with the online Paternò-Büchi reaction (HILIC-PB-MS/MS). The fatty acyl chains and C=C locations of 180 PLs molecular species, which fell into four classes, were unprecedently characterized. This exposure strategy of multi-type constituents greatly enriches the chemical profiling of PG, and helps promoting the further development of therapeutic agents and nutraceutical products from PG.

Quantitative Analysis of Methylated Adenosine Modifications Revealed Increased Levels of N 6-Methyladenosine (m6A) and N 6,2'-O-Dimethyladenosine (m6Am) in Serum From Colorectal Cancer and Gastric Cancer Patients.

Colorectal cancer and gastric cancer are the most prevalent gastrointestinal malignancies worldwide, and early detection of these cancers is crucial to reduce their incidence and mortality. RNA methylation plays an important regulatory role in a variety of physiological activities, and it has drawn great attention in recent years. Methylated adenosine (A) modifications such as N6-methyladenosine (m6A), N1-methyladenosine (m1A), 2′-O-methyladenosine (Am), N6,2′-O-dimethyladenosine (m6Am), and N6,N6-dimethyladenosine (m62A) are typical epigenetic markers of RNA, and they are closely correlated to various diseases including cancer. Serum is a valuable source of biofluid for biomarker discovery, and determination of these adenosine modifications in human serum is desirable since they are emerging biomarkers for detection of diseases. In this work, a targeted quantitative analysis method using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) was developed and utilized to analyze these methylated adenosine modifications in serum samples. The concentration differences between the healthy volunteers and cancer patients were evaluated by Mann–Whitney test, and receiver operator characteristic (ROC) curve analysis was performed to access the potential of these nucleosides as biomarkers. We demonstrated the presence of the m6Am in human serum for the first time, and we successfully quantified the concentrations of A, m6A, m1A, and m6Am in serum samples from 99 healthy controls, 51 colorectal cancer patients, and 27 gastric cancer patients. We found that the levels of m6A and m6Am in serum were both increased in colorectal cancer or gastric cancer patients, compared to that in healthy controls. These results indicate that m6A and m6Am in serum may act as potential biomarkers for early detection and prognosis of colorectal cancer and gastric cancer. In addition, the present work will stimulate investigations on the effects of adenosine methylation on the initiation and progression of colorectal cancer and gastric cancer.

A merged method for targeted analysis of amino acids and derivatives using parallel reaction monitoring combined with untargeted profiling by HILIC-Q-Orbitrap HRMS

With continuously increased scan rate and sensitivity, high resolution mass spectrometry (HRMS) allows for both reliable targeted analysis (e.g., parallel reaction monitoring, PRM) and a global overview for discovery-based untargeted profiling (e.g., data dependent acquisition, DDA) to be performed. Based on previous study on PRM for large scale targeted metabolomics quantification, we developed an innovative method merged targeted and untargeted approaches in a single run. In our workflow, the scheduled PRM for targeted analysis of amino acids and derivatives combined with the full scan was acquired in every sample injection by hydrophilic interaction liquid chromatography tandem quadrupole-Orbitrap high resolution mass spectrometry (HILIC-Q-Orbitrap HRMS). The identification of metabolic features from full scan was further performed with DDA methodology on grouped quality control (QC) samples and matched with available database. Specifically, 20 amino acids and 40 derivatives were selected for targeted analysis with optimal chromatographic separation and PRM parameters. All isomers within the selected metabolites were totally separated in the robust HILIC condition. 36 of selected metabolites were well-detected and showed a good linearity and reproducibility in NIST SRM 1950 plasma. Moreover, the absolute quantification performance of targeted PRM method was systematically validated using 10 amino acids with the corresponding stable isotope-labeled internal standards (SIL-IS). Finally, the newly developed method was successfully applied to analysis of the plasma samples from patients of pancreatic benign tumor and pancreatic cancer. The significant reduction of circulating amino acids in patients with pancreatic malignancy was confirmed by targeted PRM method and other amino acids modifications as well as polar metabolites were identified with untargeted profiling. Therefore, we have established a workflow that combines specifically and reliably targeted PRM method as well as broad-coverage untargeted profiling, which provides an innovative strategy for basic and clinical metabolomics study.

Determination of Paralytic Shellfish Toxins in Bivalve Mollusks by Amino-Modified Multiwalled Carbon Nanotube (MWCNT) Solid-Phase Extraction (SPE) and High-Performance Liquid Chromatography–Tandem Mass Spectrometry (HPLC–MS/MS)

An amino-modified multiwalled carbon nanotube (MWCNT) solid-phase extraction-based hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for simultaneous determination of 13 paralytic shellfish toxins (PSTs) in bivalve mollusks. Shellfish samples were extracted with 1% acetic acid followed by cleanup using optimized MWCNT-NH2 based solid-phase extraction. Absorbed PSTs were eluted with 2 mL 1% acetic acid–acetonitrile (70/30, v/v). The separation was performed on a Waters BEH Amide column within 7.5 min utilizing 0.1% formic acid–acetonitrile and 2 mM ammonium acetate containing 0.1% formic acid as the mobile phase. The analytes were quantified by matrix-matched external standard curves with correlation coefficients® exceeding 0.997 acquired in multiple reaction-monitoring mode. The limits of detection and quantitation in shellfish samples were from 2.76 to 9.8 μg/kg and 9.11 to 39.3 μg/kg, respectively. Recoveries at three spiked levels ranged from 71.4% to 97.8% with relative standard deviations less than 12.6%. The developed method was satisfactorily applied to the analysis of shellfish.

Kamikihito, a traditional Japanese Kampo medicine, increases the secretion of oxytocin in rats with acute stress

Ethnopharmacological relevanceKamikihito (KKT) is a Kampo medicine that is prescribed in Japan for the treatment of anemia, insomnia and mental anxiety in Japan. However, its precise mechanism of action remains unclear. Aim of the studyThis study aimed to evaluate the possible antistress effect of KKT in rats with acute stress and the contribution of oxytocin to the process. Materials and methodsAcute immobilization stress (AIS; for 90 min) was used to assess the effect of KKT on acute stress. Male Wistar rats were orally treated with KKT. Parameters of stress were evaluated, and concentrations of oxytocin in plasma and cerebrospinal fluid (CSF) were measured. ResultsAIS-induced defecation and fecal weight were significantly decreased because of treatment with KKT. The plasma levels of stress-related hormones following AIS were investigated. The pre-administration of KKT significantly increased adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) levels following AIS. Conversely, there was no significant change in the plasma oxytocin level. Microdialysis and hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) were used to monitor the oxytocin secretion in CSF. Oxytocin level increased during AIS following the treatment of KKT. At 30 min after AIS, the level remained higher than before AIS. Furthermore, using an open field test, the locomotion (exploratory behavior) immediately after AIS was examined. The total traveled distance decreased after AIS; however, the decrease was significantly inhibited by the treatment of KKT. However, the effect of KKT was obstructed by the pre-administration of the oxytocin receptor antagonist. ConclusionsThese results suggest that KKT has antistress activity and increased oxytocin secretion may be a mechanism underlying this phenomenon.