Multiple Reaction Monitoring Approach Research Articles

Evaluation of polypropylene microporous membranes as extraction devices for determination of carcinogenic aromatic amines in smoker urine by GC–MS/MS

Exposure to tobacco smoke is highly correlated to the incidence of different types of cancer due to various carcinogenic compounds present in such smoke. Aromatic amines, such as 1-naphthylamine (1-NA) and 2-naphthylamine (2-NA), are produced in tobacco burning and are linked to bladder cancer. Miniaturized solid phase extraction techniques, such as microporous membrane solid phase extraction (MMSPE), have shown potential for the extraction of aromatic compounds. In this study, a bioanalytical method for the determination of 1-NA and 2-NA in human urine was developed using polypropylene microporous membranes as a sorptive phase for MMSPE. Urine samples were hydrolyzed with HCl for 1 h at 80 °C, after which pH was adjusted to 10. Ultrasound-assisted MMSPE procedure was optimized by factorial design as follows. To each sample, 750 µL of methanol was added, and ultrasound-assisted MMSPE was conducted for 1 h with four devices containing seven 2 mm polypropylene membrane segments. After extraction, the segments were transferred to 400 µL of hexane, and desorption was conducted for 30 min. Extracts were submitted to a simple and fast microwave-assisted derivatization procedure, by the addition of 10 µL of PFPA and heating at 480 W for 3 min, followed by clean-up with phosphate buffer pH 8.0 and GC–MS/MS analysis. Adequate linearity was obtained for both analytes in a range from 25 to 500 µg L−1, while the multiple reaction monitoring approach provided satisfactory selectivity and specificity. Intra-day (n = 6) and inter-day (n = 5) precision and accuracy were satisfactory, below 15 % and between 85 and 115 %, respectively. Recovery rates found were 91.9 and 58.4 % for 1-NA and 2-NA, respectively, with adequate precision. 1-NA was found in first-hand smokers’ urine samples in a concentration range from 20.98 to 89.09 µg in 24 h, while it could be detected in second-hand smoker's urine samples, and 2-NA detected in all first and second-hand smokers’ urine samples. The proposed method expands the applicability of low cost MMSPE devices to aromatic amines and biological fluids.

Screening of polysaccharides from the differently processed products of Angelica sinensis with the best liver protection effect on chicken and the intervention mechanism study based on tandem mass tag proteomics and multiple reaction monitoring approach.

The incidence of colibacillosis in poultry is on the rise, significantly affecting the chicken industry. Ceftiofur sodium (CS) is frequently employed to treat this disease, resulting in lipopolysaccharide (LPS) buildup. Processing plays a vital role in traditional Chinese veterinary medicine. The potential intervention in liver injury by polysaccharides from the differently processed products of Angelica sinensis (PDPPAS) induced by combined CS and LPS remains unclear. This study aims to investigate the protective effect of PDPPAS on chicken liver injury caused by CS combined with LPS buildup and further identify the polysaccharides with the highest hepatoprotective activity in chickens. Furthermore, the study elucidates polysaccharides' intervention mechanism using tandem mass tag (TMT) proteomics and multiple reaction monitoring (MRM) methods. A total of 190 1-day-old layer chickens were randomly assigned into 12 groups, of which 14 chickens were in the control group and 16 in other groups, for a 10-day trial. The screening results showed that charred A. sinensis polysaccharide (CASP) had the most effective and the best hepatoprotective effect at 48 h. TMT proteomics and MRM validation results demonstrated that the intervention mechanism of the CASP high-dose (CASPH) intervention group was closely related to the protein expressions of FCER2, TBXAS1, CD34, AGXT, GCAT, COX7A2L, and CYP2AC1. Conclusively, the intervention mechanism of CASPH had multitarget, multicenter regulatory features.

A label free chemoproteomic-based platform to disclose cannabidiol molecular mechanism of action on chronic myelogenous leukemia cancer cells

The discovery of the interactome of cannabidiol (CBD), a non-psychoactive cannabinoid from Cannabis sativa L., has been here performed on chronic myelogenous leukemia cancer cells, using an optimized chemo-proteomic stage, which links Drug Affinity Responsive Target Stability with Limited Proteolysis Multiple Reaction Monitoring approaches. The obtained results showed the ability of CBD to target simultaneously some potential protein partners, corroborating its well-known poly-pharmacology activity. In human chronic myelogenous leukemia K562 cancer cells, the most fascinating protein partner was identified as the 116 kDa U5 small nuclear ribonucleoprotein element called EFTUD2, which fits with the spliceosome complex. The binding mode of this oncogenic protein with CBD was clarified using mass spectrometry-based and in silico analysis.

Unveiling the chemical components variation of Sishen formula induced by different prescription ratios by the advanced liquid chromatography/mass spectrometry approaches

Compatibility with the varying prescription ratios is a unique feature in the Chinese medicine practice. Sishen formula (SSF; containing Psoraleae Fructus-PF, Myristicae Semen-MyS, Schisandrae Chinensis Fructus-SCF, and Euodiae Fructus-EF) with different proportions has been used to treat the irritable bowel syndrome, however, the underlying chemical difference needs to be clarified, particularly the influence on extracting those known toxic components. To tackle this issue, an integrated strategy was developed: 1) systematic multicomponent characterization by an off-line two-dimensional liquid chromatography/ion mobility time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS) approach with the HDMSE-HDDDA hybrid scan; 2) discovery of potential markers associated with SSF proportion variation by untargeted metabolomics; and 3) quantitative assays of representative markers by multiple reaction monitoring (MRM) to unveil the effects on the dissolution of toxic components. Consequently, orthogonal separation, high sensitivity of detection, and intelligent data interpretation, enabled the characterization of 233 compounds from the SSF decoction. Totally 50 differential components were discovered from SSF with 16 different ratios by high-definition LC-MS profiling and multivariate statistical analysis. An MRM approach was validated offering the absolute content alternation of 18 compounds in SSF. Increasing of SCF, MyS, and EF moderately could reduce the dissolution of the main toxic coumarins of PF, while increasing of SCF promoted dissolution of the main toxic alkaloids of EF, which could be restrained by increasing MyS. Conclusively, the in-depth multicomponent characterization of SSF was achieved, and the disparities in the compatibility of various SSF proportions were exposed, which may bridge to the functions/toxicity variations of SSF in clinic.

The role of (18F)-fluoro-D-glucose positron emission tomography/computed tomography in the surveillance of abnormal myocardial energy metabolism and cardiac dysfunction in a rat model of cardiopulmonary resuscitation.

To investigate the feasibility and usefulness of 2-deoxy-2-(18F)-fluoro-D-glucose positron emission tomography/computed tomography [(18F)-FDG PET/CT] as a novel examination in the surveillance of abnormal myocardial energy metabolism and cardiac dysfunction after cardiopulmonary resuscitation (CPR). Thirteen male Sprague-Dawley rats were randomly divided into a sham group (n = 4), CPR group (n = 4), and trimetazidine (TMZ) + CPR group (n = 5). The expression levels of the myocardial injury marker cardiac troponin I (CTNI) in serum were tested at 6 hours after CPR or TMZ + CPR. The ejection fraction and fraction shortening were evaluated by echocardiography. (18F)-FDG PET/CT was used to measure the FDG uptake and the standardized uptake value (SUV) after CPR or TMZ + CPR for 6 hours. The intermediary carbohydrate metabolites of glycolysis including phosphoenolpyruvate, 3-phospho-D-glycerate, and the lactate/pyruvate ratio were detected through the multiple reaction monitoring approach. Simultaneously, the authors also tested the expression levels of the total adenosine triphosphate (ATP) and the key intermediate products of glucose ovidation as alpha ketoglutarate, citrate, and succinate in the myocardium. The authors found that the aerobic oxidation of glucose was reduced, and the anaerobic glycolysis was significantly enhanced in the myocardium in the early stage of CPR. Meanwhile, the myocardial injury marker CTNI was upregulated considerably (P = 0.014, P = 0.021), and the left ventricular function of the animal heart also markedly deteriorated with the downregulation of ATP after CPR. In contrast, myocardial injury and cardiac function were greatly improved with the increase of ATP in the CPR + TMZ group. In addition, aerobic glucose oxidation metabolites were significantly increased (P < 0.05) and anaerobic glycolysis metabolites were significantly decreased (P < 0.05) after CPR in the myocardium. Surprisingly, (18F)-FDG PET/CT could track the above changes by detecting the FDG uptake value and the SUV. Glucose metabolism is an essential factor for myocardial self-repair after CPR. (18F) FDG PET/CT, as a non-invasive technology, can monitor myocardial energy metabolism and cardiac function by tracking changes in glucose metabolism after CPR.

The identification of a potential plasma metabolite marker for Alzheimer’s disease by LC-MS untargeted metabolomics

Background and aimsAlzheimer's disease (AD), the most common type of dementia, is hard to recognize early, resulting in delayed treatment and poor outcome. At present, there is neither reliable, non-invasive methods to diagnose it accurately and nor effective drugs to recover it. Discovery and quantification of novel metabolite markers in plasma of AD patients and investigation of the correlation between the markers and AD assessment scores. Materials and methodsUntargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics with LC-quadrupole- time-of-flight (Q-TOF) was performed in plasma samples of age-matched AD patients and healthy controls. The potential markers were further quantified with targeted multiple reaction monitoring (MRM) approach. ResultsAmong the candidates, progesterone, and 3-indoleacetic acid (3-IAA) were successfully identified and then validated in 50 plasma samples from 25 AD patients and 25 matched normal controls with MRM approach. As a result, 3-IAA was significantly altered in AD patients and correlated with some AD assessment scores. ConclusionBy using untargeted LC-MS metabolomic and LC-MRM approaches to analyze plasma metabolites of AD patients and normal subjects, 3-IAA was discovered and quantified to be significantly altered in AD patients and correlated with several AD assessment scores.

Development of a new green microextraction technique, switchable hydrophilicity solvent-based homogenous liquid–liquid microextraction, to analyze synthetic cannabinoids in plasma

Synthetic cannabinoid receptor agonists (SCRA) are currently the most popular class of new psychoactive substances (NPS) in several countries, including Brazil. Notwithstanding that, they pose a threat to public health, as little of their toxicological properties is known. Moreover, these SCRAs have different presentations compared to classic cannabinoids and are not usually detected by conventional analytical methods due to their chemical variety. Thus, the development of new methodologies fit to analyze SCRAs is extremely warranted. In that regard, the use of environmentally benign or green approaches has been a trend in the last decades and has gained attention in Forensic Toxicology – so much so that guidelines have been established for that purpose, e.g. green analytical toxicology (GAT). With that in mind, the aim of this work was to develop and validate a quantitative method to analyze SCRAs in plasma by a novel green technique that agrees with the principles of GAT. A switchable hydrophilicity solvent-based homogenous liquid–liquid microextraction (SHS-HLLME) was developed and statistical tools were used to optimize it. Analysis of Variance (ANOVA) was chosen to find the best switchable hydrophilicity solvent (SHS) and Full Factorial and Central Composite design to study the following variables: volume of SHS:HCl 6 M mixture (200–500 μL) and NaOH 10 M (200–500 μL), salting-out effect, and time of extraction (1–5 min). The ANSI/ASB Standard 036, 1st edition 2019 guidelines were used for method validation. All analyses were performed using a UPLC-MS/MS with the multiple reaction monitoring approach. Ethical Committee approval No. 46404121.8.3001.0067. Funded by CNPq 142056/2020-0, CAPES/INSPEQT 16/2020, and FAPESP 21/09857-8. ANOVA showed that between two SHS tested, N,N-dimethylcyclohexylamine and dipropylamine, the latter yielded higher analyte recovery ( P < 0.05). Moreover, the Design of Experiment tools herein used showed that the salting-out effect and time of extraction had no major impact on analyte recovery; in contrast, 500 μL of both SHS:HCl mixture and NaOH yielded higher analyte recovery ( P < 0.05). Once optimized, the proposed technique was successfully validated according to international guidelines with the following parameters: limits of detection: 0.01–0.08 ng/mL; limit of quantitation: 0.1 ng/mL; linearity: 0.1–10 ng/mL; bias and precision < 15%; no carryover was observed and the method proved to be selective for the aimed SCRAs. The matrix effect, recovery efficiency, and process efficiency studies are being currently carried out. The microextraction technique herein developed is based on an acid-base neutralization between HCl and NaOH, making it an approach significantly more sustainable when compared to conventional choices that rely entirely on organic solvents. In that regard, only 250 μL of dipropylamine was required–an organic solvent considerably less toxic than those used in common extractions, organochlorides for example. In addition, a small volume was used compared to more conventional techniques in forensic analyses, such as solid-phase extraction (> 500 μL). Hence, we were able to successfully detect and quantitate 31 SCRAs at extremely low concentrations (0.01–0.1 ng/mL, respectively) using only 300 μL of plasma, showing that the SHS-HLLME can be used for the analysis of multiple SCRAs in a single run, thus could be a valuable tool in forensic cases. We demonstrated for the first time the application of the novel SHS-HLLME for synthetic cannabinoids. The microextraction technique herein developed has promising applications in Forensic Toxicology, as it consists of a simple and fast procedure that yields high sensitivity with only a few microliters of a biological sample. Also, it avoids the use of highly toxic chemicals and generates less residue, thus it fits the growing trend of using sustainable analytical methodologies, established by GAT and other similar guidelines.

Prognosis of Alzheimer's Disease Using Quantitative Mass Spectrometry of Human Blood Plasma Proteins and Machine Learning.

Early recognition of the risk of Alzheimer’s disease (AD) onset is a global challenge that requires the development of reliable and affordable screening methods for wide-scale application. Proteomic studies of blood plasma are of particular relevance; however, the currently proposed differentiating markers are poorly consistent. The targeted quantitative multiple reaction monitoring (MRM) assay of the reported candidate biomarkers (CBs) can contribute to the creation of a consistent marker panel. An MRM-MS analysis of 149 nondepleted EDTA–plasma samples (MHRC, Russia) of patients with AD (n = 47), mild cognitive impairment (MCI, n = 36), vascular dementia (n = 8), frontotemporal dementia (n = 15), and an elderly control group (n = 43) was performed using the BAK 125 kit (MRM Proteomics Inc., Canada). Statistical analysis revealed a significant decrease in the levels of afamin, apolipoprotein E, biotinidase, and serum paraoxonase/arylesterase 1 associated with AD. Different training algorithms for machine learning were performed to identify the protein panels and build corresponding classifiers for the AD prognosis. Machine learning revealed 31 proteins that are important for AD differentiation and mostly include reported earlier CBs. The best-performing classifiers reached 80% accuracy, 79.4% sensitivity and 83.6% specificity and were able to assess the risk of developing AD over the next 3 years for patients with MCI. Overall, this study demonstrates the high potential of the MRM approach combined with machine learning to confirm the significance of previously identified CBs and to propose consistent protein marker panels.

Isolation, method development, and validation of diterpenes in coffee oil: identification by multiple reaction monitoring approach with atmospheric pressure chemical ionization mode

Pharmacognosy Research,2021,13,1,42-48.DOI:10.4103/pr.pr_66_20Published:April 2021Type:Original ArticleAuthors:Naveen Puttaswamy, Lingaraju Harakanahalli Basavegowda, Deepak Machettira, Chandrappa Siddappa, Rashmi Ramesh, and Shyam Prasad Kodimule Author(s) affiliations:Naveen Puttaswamy1, Lingaraju Harakanahalli Basavegowda2, Deepak Machettira2, Chandrappa Siddappa2, Rashmi Ramesh1, Shyam Prasad Kodimule2 1Analytical Research and Development, Vidya Herbs Pvt. Ltd, Bengaluru, Karnataka, INDIA 2Phytochemistry Lab, Vidya Herbs Pvt. Ltd., Bengaluru, Karnataka, INDIA Abstract:Objectives: The aim of the present study is to isolation, identification, and quantification of free diterpenes in coffee oil of Coffea arabica. Materials and Methods: For the isolation of free diterpenes, saponification reaction step was employed and followed with a liquid-liquid extraction procedure applied. Cafestol and kahweol were identified by multiple reaction monitoring (MRM) of positive ionization (MRM) mode of atmospheric pressure chemical ionization (APCI) mass spectrometry detection. The quantification of free diterpenes was done by high-performance liquid chromatography with diode array detector. The developed method was validated according to the international conference on harmonization guidelines. Results: The ion transitions of the precursor to the product ion at (M + H)+ m/z 317.20→146.90, 281.00, 299.00 for cafestol and 315.10→144.90, 278.90, 296.90 for kahweol were observed. The proposed method was validated for linearity with excellent correlation coefficient of cafestol and kahweol were found to be 0.9997 and 0.9996, respectively. The intra-day and intermediate precisions and repeatability showed the percentage relative standard deviation was <1%. The recovery rate for cafestol and kahweol were observed to be in the acceptable limit of 95.303%–98.539% and 95.963%–97.174%, respectively. The limit of detection of cafestol and kahweol were found to be 6.81 and 7.35 ppm, respectively. The limit of quantitation (LOQ) was found to be 22.72 and 24.52 ppm, respectively. Conclusion: The developed method is simple, rapid, precise, accurate and it is recommended for efficient assays in routine work. Keywords:Atmospheric pressure chemical ionization, Cafestol, Coffea arabica, Kahweol, Method validationView:PDF (1.44 MB)

Mass Spectrometric Analysis of Bioactive Sphingolipids in Fungi.

As biomolecules, sphingolipids represent a broad spectrum of structures ranging from simple long chain bases to complex glycosphingolipids. While several different mass spectrometry based approaches have been proven to be useful in qualitative and quantitative analysis of sphingolipids, we find that electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the multiple-reaction monitoring (MRM) mode using a triple quadrupole instrument, coupled to high-performance liquid chromatography (HPLC), is the most suitable approach for the analysis. In this chapter, we describe the method in a step-by-step manner towards the targeted analysis of sphingolipids in fungi. With optimized HPLC separation and instrument settings, this MRM approach affords detection of many sphingolipid species simultaneously with good sensitivity.

Evaluating a targeted multiple reaction monitoring approach to global untargeted lipidomic analyses of human plasma.

The Lipidyzer platform was recently updated on a SCIEX QTRAP 6500+ mass spectrometer and offers a targeted lipidomics assay including 1150 different lipids. We evaluated this targeted approach using human plasma samples and compared the results against a global untargeted lipidomics method using a high-resolution Q Exactive HF Orbitrap mass spectrometer. Lipids from human plasma samples (N = 5) were extracted using a modified Bligh-Dyer approach. A global untargeted analysis was performed using a Thermo Orbitrap Q Exactive HF mass spectrometer, followed by data analysis using Progenesis QI software. Multiple reaction monitoring (MRM)-based targeted analysis was performed using a QTRAP 6500+ mass spectrometer, followed by data analysis using SCIEX OS software. The samples were injected on three separate days to assess reproducibility for both approaches. Overall, 465 lipids were identified from 11 lipid classes in both approaches, of which 159 were similar between the methods, 168 lipids were unique to the MRM approach, and 138 lipids were unique to the untargeted approach. Phosphatidylcholine and phosphatidylethanolamine species were the most commonly identified using the untargeted approach, while triacylglycerol species were the most commonly identified using the targeted MRM approach. The targeted MRM approach had more consistent relative abundances across the three days than the untargeted approach. Overall, the coefficient of variation for inter-day comparisons across all lipid classes was ∼ 23% for the untargeted approach and ∼ 9% for the targeted MRM approach. The targeted MRM approach identified similar numbers of lipids to a conventional untargeted approach, but had better representation of 11 lipid classes commonly identified by both approaches. Based on the separation methods employed, the conventional untargeted approach could better detect phosphatidylcholine and sphingomyelin lipid classes. The targeted MRM approach had lower inter-day variability than the untargeted approach when tested using a small group of plasma samples. These studies highlight the advantages in using targeted MRM approaches for human plasma lipidomics analysis.

Melatonin down-regulates steroidal hormones, thymocyte apoptosis and inflammatory cytokines in middle-aged T. cruzi infected rats

Chagas disease, triggered by the flagellate protozoan Trypanosoma cruzi (T. cruzi) plays a potentially threat to historically non-endemic areas. Considerable evidence established that the immuno-endocrine balance could deeply influence the experimental T. cruzi progression inside the host's body. A high-resolution multiple reaction monitoring approach (MRMHR) was used to study the influence of melatonin on adrenal and plasma steroidal hormones profile of T. cruzi infected Wistar rats. Young (5 weeks) and middle-aged (18 months) male Wistar rats received melatonin (5 mg/Kg, orally) during the acute Chagas disease. Corticosterone, 11-dehydrocorticosterone (11-DHC), cortisol, cortisone, aldosterone, progesterone and melatonin concentration were evaluated. Interleukin-1 alpha and β (IL-1α and β), IL-6 and transforming growth factor beta (TGF-β) were also analyzed. Our results revealed an increased production of corticosterone, cortisone, cortisol and aldosterone in middle-aged control animals, thus confirming the aging effects on the steroidal hormone profile. Serum melatonin levels were reduced with age and predominantly higher in young and middle-aged infected rats. Melatonin treatment reduced the corticosterone, 11-DHC, cortisol, cortisone, aldosterone and progesterone in response to T. cruzi infection. Decreased IL-1 α and β concentrations were also found in melatonin treated middle-aged infected animals. Melatonin treated middle-aged control rats displayed reduced concentrations of TGF-β. Melatonin levels were significantly higher in all middle-aged rats treated animals. Reduced percentages of early and late thymocyte apoptosis was found for young and middle-aged melatonin supplemented rats. Finally, our results show a link between the therapeutic and biological effects of melatonin controlling steroidal hormones pathways as well as inflammatory mediators.

Stilbenes in grapes and wines of Tannat, Marselan and Syrah from Uruguay

Aim: The aim of the study was to investigate the stilbene composition of grapes and wines of the Vitis vinifera cultivars Tannat, Marselan and Syrah cultivated in Uruguay. The effects of delaying the harvest on stilbene concentrations were determined, and the stability of stilbenes during wine storage was assessed.Methods and results: Stilbene concentrations were determined in the grapes and wines of two vintages (2015 and 2016) and two harvest dates for each cultivar. Vinification was carried out by traditional maceration, and samples of the wines of each vintage were analysed in the period from 3 months after devatting to up to 24 months later. After solid-phase extraction, stilbenes were identified and quantified by HPLC-ESI-MS/MS using a multiple reaction-monitoring approach. In the grape berries, stilbene concentrations were between 1.6 and 7.7 mg/kg, depending on grape cultivar, growing season, and in Syrah, harvest date. In the wines, stilbene concentrations were initially between 0.9 and 5.0 mg/L, being highest in Syrah, lowest in Marselan, and intermediate in Tannat. Stilbene concentrations in the Marselan wines were lower than expected based on stilbene concentrations in the grapes from which they were produced, suggesting poor extraction during winemaking. Total stilbene concentrations remained very stable during the analytical period.Conclusions: Delaying the harvest does not necessarily increase the stilbene content of grapes, but it can do so significantly, as shown for Syrah. For some grape cultivars, such as Marselan, poor extraction of stilbenes during winemaking can limit their concentrations in the resulting wines.Significance and impact of the study: The results of this study show the relevance of grape cultivar, degree of maturity and storage time may have into stilbenes. They provide reference data on the stilbene composition of grapes and wines produced under Uruguayan winegrowing conditions. The high stability of stilbenes during wine storage is relevant for consumers interested in red wine as a source of bioactive compounds.&#x0D; In memory of Prof. Isidro Hermosín-Gutiérrez.

An ultra-robust fingerprinting method for quality assessment of traditional Chinese medicine using multiple reaction monitoring mass spectrometry

Chromatographic fingerprinting has been perceived as an essential tool for assessing quality and chemical equivalence of traditional Chinese medicine. However, this pattern-oriented approach still has some weak points in terms of chemical coverage and robustness. In this work, we proposed a multiple reaction monitoring (MRM)-based fingerprinting method in which approximately 100 constituents were simultaneously detected for quality assessment. The derivative MRM approach was employed to rapidly design MRM transitions independent of chemical standards, based on which the large-scale fingerprinting method was efficiently established. This approach was exemplified on QiShenYiQi Pill (QSYQ), a traditional Chinese medicine-derived drug product, and its robustness was systematically evaluated by four indices: clustering analysis by principal component analysis, similarity analysis by the congruence coefficient, the number of separated peaks, and the peak area proportion of separated peaks. Compared with conventional ultraviolet-based fingerprints, the MRM fingerprints provided not only better discriminatory capacity for the tested normal/abnormal QSYQ samples, but also higher robustness under different chromatographic conditions (i.e., flow rate, apparent pH, column temperature, and column). The result also showed for such large-scale fingerprints including a large number of peaks, the angle cosine measure after min-max normalization was more suitable for setting a decision criterion than the unnormalized algorithm. This proof-of-concept application gives evidence that combining MRM technique with proper similarity analysis metrices can provide a highly sensitive, robust and comprehensive analytical approach for quality assessment of traditional Chinese medicine.

Sensitive Determination of C-Alkylated Flavonoids by HPLC-ESI-MS/MS Using Multiple Reaction Monitoring Approach: Pseudarthria hookeri as a Case Study.

One of the major problems with the formulation of herbal medicines is the quality control of plant material to ensure its efficacy and safety. Quality control of medicinal plants requires analysis of many bioactive compounds present in the plant. C-alkylated flavonoids are an important bioactive subclass of flavonoids. A simple, rapid, sensitive and selective method is presented here for the quantification of bioactive C-alkylated flavonoids. This is the first quantitative method for analysis of C-alkylated flavonoids based on the multiple reaction monitoring (MRM) approach so far. This study focuses on method development for quantification of bioactive C-alkylated flavonoids. Quantification of a total of five C-alkylated flavonoids was done employing the MRM approach on an HPLC-QqQ-MS instrument. LODs and LOQs for quantified flavonoids were in the range of 0.41-1.32 and 1.23-3.96ng/mL, respectively. Linear calibration curves between 25 and 1500ng/mL were obtained with the regression coefficients of ≥0.996. Accuracy (% bias) and precision (% RSD) of the analyses were found to be less than 5%. Developed HPLC-ESI-MS/MS can be employed as a quality control method of plant raw materials.

Decreased Amino Acid Concentrations are Involved in Congenital Heart Disease

Objective: Congenital heart disease (CHD) is the most common malformation in China. In this study, we determined whether amino acids (AAs) in the amniotic fluid (AF) of patients with CHD changed and clarified whether AAs would affect the insulin-like growth factor type 1 receptor (IGF1R). Method: Fifty-seven AF samples from pregnant women carrying CHD-affected (n = 17) or normal (n = 40) fetuses were collected. The AA concentrations were measured in AF by liquid chromatography-tandem mass spectrometry. The IGF axis-related and epigenetic marker proteins in AF after serial treatments were quantified using a multiple reaction monitoring approach. IGF1R and P300 were also confirmed by Western blot in AF without any treatment. Results: Most AAs decreased in the AF of patients with CHD. P300 and IGF1R decreased significantly in the CHD group. When H9C2 cells were cultured in one-half AA concentrations, the expression of P300 and IGF1R was reduced. Histone acetylation of the IGF1R promoter also decreased. Conclusion: Our data suggest that AAs decreased in the AF of patients with CHD. AAs may partly regulate the IGF1R through P300, which may be involved in heart development.

Label-free targeted LC-ESI-MS2 analysis of human milk oligosaccharides (HMOS) and related human milk groups with enhanced structural selectivity.

Human milk (HM) supports the healthy development of neonates and exerts many of its beneficial effects via contained free human milk oligosaccharides (HMOS). These HMOS exhibit a complexity and structural diversity that pose a significant analytical challenge. A detailed characterization of HMOS is essential as every individual structure may have a different function/activity. Certain HMOS isomers may even fundamentally differ in their biological function, and especially their characterization by LC or LC-MS is often impaired by co-elution phenomena. Thus, more efficient analytical methodologies with enhanced structural selectivity are required. Therefore, we developed a negative ion mode LC-ESI-MS2 approach featuring straightforward sample preparation, environmentally friendly EtOH gradient elution, and enhanced, semiquantitative characterization of distinct native HMOS by multiple reaction monitoring (MRM). Our MRM-LC-MS setup takes advantage of highly selective, glycan configuration-dependent collision-induced dissociation (CID) fragments to identify individual neutral and acidic HMOS. Notably, many human milk oligosaccharide isomers could be distinguished in a retention time-independent manner. This contrasts with other contemporary MRM approaches relying on rather unspecific MRM transitions. Our method was used to determine the most abundant human milk tri-, tetra-, penta-, and hexaoses semiquantitatively in a single LC-MS assay. Detected HMO structures included fucosyllactoses (e.g., 2'-FL), lacto-N-difucotetraose (LDFT), lacto-N-tetraoses (LNTs), lacto-N-fucopentaoses (e.g., LNFP I, LNFP II and III), lacto-N-difucohexaoses (LNDFHs) as well as sialyllactoses (SLs) and tentatively assigned blood group A and B tetrasaccharides from which correct human milk type assignment could be also demonstrated. Correctness of milk typing was validated for milk groups I-IV by high pressure anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (HPAEC-PAD). Graphical Abstract ᅟ.

Microfluidics-based liquid chromatography/mass spectrometry multiple reaction monitoring approach for the relative quantification of Burkholderia cenocepacia secreted virulence factors.

RationaleBurkholderia cenocepacia is an opportunistic pathogen that is commonly isolated from patients with cystic fibrosis (CF). Quorum sensing has been suggested to play a role in the activity of type II and type VI secretion systems and the release of virulence factors. Apart from the classical acyl homoserine lactone quorum sensing, B. cenocepacia also uses the diffusible signal factor system (DSF). Quantitative information on the true impact of DSF molecules on the release of ZmpA and other virulence factors is lacking.MethodsBased on results of a label‐free proteomics analysis addressing changes in the secretome in response to DSFs, a panel of peptides was selected to develop a microfluidics liquid chromatography/mass spectrometry (LC/MS) method implementing single reaction monitoring (SRM) to quantify B. cenocepacia virulence factors.ResultsIncrease in secretion of virulence factors upon treatment with BDSF was observed for ZmpA and Aida, but not for ZmpB. Type VI secretion system dependent Hcp1 and TecA were decreased. However, non‐physiological amounts of BDSF were needed to provoke the effect. DSFs from P. aeruginosa and S. maltophilia were also affecting virulence factor secretion, but the effect was smaller than for the endogenous BDSF.ConclusionsMicrofluidics‐based SRM is a useful tool to quantitatively assess the impact of quorum sensing on the release of virulence factors by (opportunistic) pathogens.

A new mass spectrometry-based method for the quantification of histones in plasma from septic shock patients

The aim of this study was to develop a novel method to detect circulating histones H3 and H2B in plasma based on multiple reaction monitoring targeted mass spectrometry and a multiple reaction monitoring approach (MRM-MS) for its clinical application in critical bacteriaemic septic shock patients. Plasma samples from 17 septic shock patients with confirmed bacteraemia and 10 healthy controls were analysed by an MRM-MS method, which specifically detects presence of histones H3 and H2B. By an internal standard, it was possible to quantify the concentration of circulating histones in plasma, which were significantly higher in patients, and thus confirmed their potential as biomarkers for diagnosing septic shock. After comparing surviving patients and non-survivors, a correlation was found between higher levels of circulating histones and unfavourable outcome. Indeed, histone H3 proved a more efficient and sensitive biomarker for septic shock prognosis. In conclusion, these findings suggest the accuracy of the MRM-MS technique and stable isotope labelled peptides to detect and quantify circulating plasma histones H2B and H3. This method may be used for early septic shock diagnoses and for the prognosis of fatal outcomes.

LC-MS/MS analysis of lipidized analogs of prolactin-releasing peptide utilizing a monolithic column and simple sample preparation.

Novel compounds for obesity treatment are currently being studied employing lipidized analogs of anorexigenic neuropeptides. Various analogs of prolactin-releasing peptide have demonstrated their ability to decrease food intake. Adequate analytical tools are required to support corresponding research. Methodology & results: An analytical method was developed that includes simple dilution of plasma samples prior to liquid chromatography-mass spectrometry and employs a monolithic column for the determination of lipidized analogs of prolactin-releasing peptide in complex biological samples. A multiple reaction monitoring approach was applied that included matrix calibration and an internal standard and produced a linear calibration range 20-200ng ml-1 in rat and macaque plasma samples. A straightforward, simple and reliable analytical method was developed satisfying major validation criteria.