Liquid Chromatography-triple Quadrupole Tandem Mass Research Articles

Prediction of Hopeless Peptides Unlikely to be Selected for Targeted Proteome Analysis.

In targeted proteomics using liquid chromatography-tandem triple quadrupole mass spectrometry (LC/MS/MS) in the selected reaction monitoring (SRM) mode, selecting the best observable or visible peptides is a key step in the development of SRM assay methods of target proteins. A direct comparison of signal intensities among all candidate peptides by brute-force LC/MS/MS analysis is a concrete approach for peptide selection. However, the analysis requires an SRM method with hundreds of transitions. This study reports on the development of a method for predicting and identifying hopeless peptides to reduce the number of candidate peptides needed for brute-force experiments. Hopeless peptides are proteotypic peptides that are unlikely to be selected for targets in SRM analysis owing to their poor ionization characteristics. Targeted proteomics data from Escherichia coli demonstrated that the relative ionization efficiency between two peptides could be predicted from sequences of two peptides, when a multivariate regression model is used. Validation of the method showed that >20% of the candidate peptides could be successfully eliminated as hopeless peptides with a false positive rate of less than 2%.

Simple extraction method for quantification of phenothiazine residues in pork muscle using liquid chromatography-triple quadrupole tandem mass spectrometry.

In this study, an analytical method was developed for quantification of residues of the anthelmintic drug phenothiazine (PTZ) in pork muscle using liquid chromatography-tandem mass spectrometry. Muscles were extracted using 0.2% formic acid and 10 mm ammonium formate in acetonitrile, defatted and purified using n-hexane. The drug was well separated on a Waters XBridge™ C18 analytical column using a binary solvent system consisting of 0.2% formic acid and 10 mm ammonium formate in ultrapure water (A) and acetonitrile (B). Good linearity was achieved over a six-point concentration range in matrix-matched calibration with determination coefficient =0.9846. Fortified pork muscle having concentrations equivalent to and double the limit of quantification (1 ng/g) yielded recovery ranges between 100.82 and 104.03% and relative standard deviations <12%. Samples (n = 5) collected from large markets located in Seoul City tested negative for PTZ residue. In conclusion, 0.2% formic acid and ammonium formate in acetonitrile can effectively extract PTZ from pork muscle without solid-phase extraction, a step normally required for cleanup before analysis and the validated method can be used for routine analysis to ensure the quality of animal products.

Phytohormone Profiling During Tuber Development of Chinese Yam by Ultra-high performance Liquid Chromatography–Triple Quadrupole Tandem Mass Spectrometry

Changes in agronomic characters and the profile of various endogenous phytohormones during tuber development were studied in Dioscorea opposite (Chinese yam) cv. Guihuai 16. Tuber development exhibited a sigmoidal growth pattern according to the changes in tuber agronomic characters. The growth cycle of yam tuber could be divided into three stages: initiation stage, enlargement stage, and maturation stage. Moreover, the enlargement stage could be separated into three phases—slow growth phase, rapid growth phase, and late growth phase. Endogenous changes in phytohormones were associated with developmental changes in the tubers. The pulses of bioactive gibberellins (such as GA3 and GA4) were measured in tubers. The highest contents of GA3 and GA4 were reached 90 days after field planting, corresponding to the beginning of the rapid growth phase of tuber enlargement. Changes in trans-zeatin (tZ), jasmonic acid (JA), indole-3-acetic acid (IAA), and abscisic acid (ABA) levels were also observed, and seemed to be related to tuber enlargement at different phases. Continuous increases in JA and tZ contents accompanied tuber enlargement. Transient pulses of both IAA and ABA contents were also observed at the start of tuber rapid growth. Additionally, a second peak level of IAA was detected at the tuber maturation stage. These results suggest GAs play a key role at the beginning of the tuber rapid growth stage, and there is a close relationship between whole tuber enlargement and the contents of JA and tZ. Moreover, it is suggested that IAA and ABA also may be linked to the beginning of tuber rapid growth, and IAA also seems to be correlated to late tuber maturation.

Pharmacokinetics and pharmacodynamics study of rhein treating renal fibrosis based on metabonomics approach

BackgroundThe selection of effect indicators in the pharmacokinetic/ pharmacodynamic study of complex diseases to describe the relationship between plasma concentration and effect indicators is difficult. PurposeThree effect indicators of renal fibrosis were successfully determined. The relationship between pharmacokinetics and pharmacodynamics of rhein in rhubarb was elucidated. Study designThe study was a metabolomics analysis of rat plasma and pharmacokinetics/ pharmacodynamics of rhein. MethodsA sensitive and simple ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-MS/MS) method was applied to determine the rhein plasma concentration in the rat model of renal fibrosis and rat sham-operated group after the administration of rhubarb decoction. Then, the ultra performance liquid chromatography-Micromass quadrupole-time of flight mass spectrometry (UPLC-QTOF/MS) metabolomics method was used to screen biomarkers of renal fibrosis in rat plasma. Furthermore, the relationship between the plasma concentration of rhein and the concentration of three biomarkers directly related to renal fibrosis were analyzed. ResultsThe three screened biomarkers could represent the effect of rhein treatment on renal fibrosis. Increasing the plasma concentration of rhein tended to restore the concentration of the three biomarkers in the model group compared with that in the sham-operated group. Evident differences in the area under the plasma concentration-time curve (AUC) of rhein were also observed under different pathological states. The results provide valuable information for the clinical application of rhubarb. ConclusionRhein intervention could recover the physiological balance in living organisms from the pharmacokinetic/pharmacodynamic levels. New information on the pharmacokinetic/pharmacodynamic study of complex diseases is provided.

Determination of illicit drugs in aqueous environmental samples by online solid-phase extraction coupled to liquid chromatography–tandem mass spectrometry

In this study, a fully automated analytical method, based on online solid phase extraction coupled to liquid chromatography-triple quadrupole tandem mass spectrometry (online SPE-LC-MS/MS), has been developed and optimized for the quantification of 10 illicit drugs and metabolites in environmentally aqueous samples collected from China. The particular attention was devoted to minimize the matrix effects through a washing step, which washed out the interferences effectively and helped to reduce the matrix effect significantly. The key advantages of the method are high sensitivity, selectivity and reliability of results, smaller sample manipulation, full automation, and fairly high throughput. The whole procedure was then successfully applied in the analysis of various surface water and wastewater effluents samples. Pseudoephedrine have been detected at trace levels (several tens ng L−1 or less), while MDA, MDMA, benzoylecgonine and methadone were below the LOQ in all samples. Caffeine, cotinine and paraxanthine, which may be derived from medicines and foods, were detected with the highest frequencies and concentrations.

Simultaneous Determination of Nine Plant Growth Regulators in Navel Oranges by Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry

A new and reliable method for the simultaneous determination of nine plant growth regulators in navel oranges was developed by liquid chromatography-triple quadrupole tandem mass (QqQ LC/MS). Extraction was performed by acetone and ethyl acetate (v/v, 1:2) with microwave-assisted extraction technique. Cleanup of extracts was performed with dispersive-solid phase extraction (d-SPE) using active carbon as the sorbent. The identification of each analyte was established by chromatographic retention time, analyte-specific fragmentation patterns, and relative peak area ratios of precursor/production pairs. Under the optimized conditions, the average recoveries (six replicates), except for methyl naphthacetate, were in the range of 61.0–109.4 % and limits of detection ranging from 0.01 to 162.2 μg kg−1. The results demonstrated that the developed QqQ LC/MS, extraction and purification method is highly effective for analyzing trace amounts of target plant growth regulators (PGRs) in navel orange samples.

Determination of RH-5849 and indoxacarb in rice straw, rice husk, brown rice and soil using liquid chromatography-tandem triple quadrupole mass spectrometry following extraction with QuEChERS method.

A fast and simple analytical method was developed for the simultaneous determination of RH-5849 and indoxacarb in soil, rice straw, rice husk and brown rice. QuEChERS (quick, easy, cheap, effective, rugged and safe) method was used for extraction, and liquid chromatography with tandem triple quadrupole mass spectrometry was used for quantification. The matrix-matched calibration plots were linear in the range between 25 and 5000 μg/L for soil, rice straw, rice husk and brown rice samples. All determination coefficients (R(2) ) were ≥0.9962. The limits of detection and quantification were 1.5 and 5 μg/kg, respectively. Recoveries at three fortification levels ranged between 79.5 and 97.9% with relative standard deviations <11%. The developed method was validated and applied for the analysis of dissipation study samples. For field experiments, the half-lives of RH-5849 and indoxacarb in rice straw were 11.93 and 5.83 days, respectively. The method was demonstrated to be reliable for the routine monitoring of RH-5849 and indoxacarb in rice samples. Copyright © 2016 John Wiley & Sons, Ltd.

Determination of Anabolic Hormones in Porcine Hair by High-Performance Liquid Chromatography–Mass Spectrometry

ABSTRACTA sensitive, rapid, efficient, and reliable method is reported for the determination of testosterone, 17α-methyltestosterone, trenbolone, trenbolone acetate, boldenone, nandrolone phenylpropionate, nandrolone, and norethindrone in porcine hair. This protocol involves treating 100 mg of pulverized hair by mild digestion using tris(2-carboxyethyl)phosphine hydrochloride and subsequent extraction of the anabolic hormones with methyl tert-butyl ether. The separation of the analytes was performed using acetonitrile and 0.1% aqueous formic acid. Quantitative analysis was performed by high-performance liquid chromatography–tandem triple-quadrupole mass spectrometry with positive electrospray ionization. This method is accurate, offers good sensitivity and high recovery, allowing the determination of anabolic hormones in porcine hair.

Degradation kinetics and pathways of spirotetramat in different parts of spinach plant and in the soil.

Spirotetramat is a new pesticide against a broad spectrum of sucking insects and exhibits a unique property with a two-way systemicity. In order to formulate a scientific rationale for a reasonable spray dose and the safe interval period of 22.4% spirotetramat suspension concentrate on controlling vegetable pests, we analyzed degradation dynamics and pathways of spirotetramat in different parts of spinach plant (leaf, stalk, and root) and in the soil. We conducted experimental trials under field conditions and adopted a simple and reliable method (dispersive solid phase extraction) combined with liquid chromatography-triple quadrupole tandem mass spectrometry to evaluate the dissipation rates of spirotetramat residue and its metabolites. The results showed that the spirotetramat was degraded into different metabolite residues in different parts of spinach plant (leaf, stalk, and root) and in the soil. Specifically, spirotetramat was degraded into B-keto, B-glu, and B-enol in the leaf; B-glu and B-enol in the stalk; and only B-enol in the root. In the soil where the plants grew, spirotetramat followed a completely different pathway compared to the plant and degraded into B-keto and B-mono. Regardless of different degradation pathways, the dissipation dynamic equations of spirotetramat in different parts of spinach plant and in the soil were all based on the first-order reaction dynamic equations. This work provides guidelines for the safe use of spirotetramat in spinach fields, which would help prevent potential health threats to consumers.

Simple extraction method requiring no cleanup procedure for the detection of minocycline residues in porcine muscle and milk using triple quadrupole liquid chromatography-tandem mass spectrometry

A versatile analytical method was developed for simple detection of minocycline residues in porcine muscle and milk using liquid chromatography-triple quadrupole tandem mass spectrometry (LC/MS/MS) with electrospray ionization. Samples were extracted with a mixture of acetonitrile and ethyl acetate (2:1, v/v), and then defatted using n-hexane. No cleanup procedure was deemed necessary. Minocycline was separated on a reversed-phase analytical column using a combination of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B) as the mobile phase. Matrix-matched calibration showed good linearity over a concentration range of 10–60 μg/kg with a determination coefficient (R 2 ) of 0.9727. Fortified porcine and milk samples having concentrations equivalent to and double the limit of quantification (LOQ = 10 ng/g), respectively, yielded recovery ranges between 83.02 and 8.03 % and relative standard deviations <18 %. Samples collected from a large market located in Seoul, Korea, tested negative for minocycline residue. These results show that a combination of acetonitrile and ethyl acetate can effectively extract minocycline from porcine muscle and milk without solid-phase extraction, a step usually required for cleanup before analysis. The developed method is simple, sensitive, and can be extrapolated to other food animal products that are rich in protein and fats.

Effects of ZnO Nanoparticle on Photo-Protection and Insecticidal Synergism of Rotenone

&lt;p&gt;Rotenone has an effective insecticidal activity. However, the photodegradation of rotenone under sunlight or UV (ultraviolet light) leads to negative effects on its insecticidal activity and persistence. This study examined the photo-protection of rotenone when exposed to UV combinated with various nanoparticles. The remaining concentration of rotenone was analyzed by LC-MS/MS (liquid chromatography-triple quadrupole tandem mass spectrometry) at particular intervals. It indicated that various nanoparticles had different effects and combination with ZnO nanoparticle provided remarkable degree of photo-protection of rotenone in UV radiation. In comparision with ZnO, SiO&lt;sub&gt;2 &lt;/sub&gt;nanoparticle provided moderate degree of photo-protection of rotenone in UV radiation. In addition, TiO&lt;sub&gt;2&lt;/sub&gt;, Fe&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3 &lt;/sub&gt;and CuO nanoparticle exerted catalytic degradation effects on rotenone to a certain degree.The combination of rotenone and ZnO nanoparticle(4:1) increased the effciency of mortality to the highest compared with the same concentration of sole rotenone or ZnO nanoparticle treatment alone and their co-toxicity coefficient was 128.63. ZnO nanoparticle has good UV photo-protective properties and insecticidal synergism on rotenone. The application of this proposed method can provide significant and practical guidance for improving the photostability and insecticidal activity of rotenone as well as other biopesticides.&lt;/p&gt;

Use of designed experiments for the improvement of pre-analytical workflow for the quantification of intracellular nucleotides in cultured cell lines

The present study is focused on the development of a pre-analytical strategy for the quantification of intracellular nucleotides from cultured cell lines. Different protocols, including cell recovery, nucleotide extraction and purification, were compared on a panel of nucleoside mono-, di- and triphosphates from four cell lines (adherent and suspension cells). The quantification of nucleotides was performed using a validated technique with on-line solid-phase extraction coupled with liquid chromatography–triple quadrupole tandem mass spectrometry (LC–MS/MS). Designed experiments were implemented to investigate, in a rigorous and limited-testing experimental approach, the influence of several operating parameters. Results showed that the technique used to harvest adherent cells drastically affected the amounts of intracellular nucleotides. Scraping cells was deleterious because of a major leakage (more than 70%) of intracellular nucleotides during scraping. Moreover, some other tested conditions should be avoided, such as using pure methanol as extraction solvent (decrease over 50% of intracellular nucleotides extracted from NCI-H292 cells) or adding a purification step with chloroform. Designed experiments allowed identifying an interaction between the percentage of methanol and the presence of chloroform. The mixture methanol/water (70/30, v/v) was considered as the best compromise according to the nucleoside mono-, di-, or triphosphates and the four cell lines studied. This work highlights the importance of pre-analytical step combined with the cell lines studied associated to sensitive and validated assay for the quantification of nucleotides in biological matrices.

Pharmacokinetic profile and metabolite identification of yuanhuapine, a bioactive component in Daphne genkwa by ultra-high performance liquid chromatography coupled with tandem mass spectrometry

Daphne genkwa Sieb. et Zucc. (Thymelaeaceae) is mainly used for the treatment of edema, asthma, and cancer in China and Korea for centuries. The major bioactive components in D. genkwa are daphnane-type diterpenoids, which showed pharmacological activities such as antileukemic, antifertility and skin irritants. In this study, the pharmacokinetics and metabolism profile of yuanhuapine, an effective and toxic diterpenoid, was investigated in rats. The plasma exposure of yuanhuapine was determined by ultra-performance liquid chromatography tandem triple-quadrupole mass spectrometry (UPLC–TQ–MS), and the pharmacokinetic parameters were calculated using the DAS 2.0 pharmacokinetic program. The metabolites were identified through ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC–Q–TOF/MS) and a Metabolynx™ (v4.1) program. After oral administration (5mg/kg), yuanhuapine was slowly absorbed and reached a maximum concentration of 579.20±212.85ng/mL at 7.33±1.03h, it also eliminated slowly. As the cumulative excretion of yuanhuapine in urine and feces were only 0.7% and 3.3%, we supposed that biotransformation in vivo was of significant importance to this component. Not only the prototype but also twelve metabolites were found and tentatively identified in rat urine after oral administration of yuanhuapine. The metabolic pathway mainly involves hydroxylation, methylation, glucuronidation and cysteine conjugation during the phase I and phase II biotransformation pathway. All the information gained here was useful in understanding the pharmacological actions and toxic properties of yuanhuapine, and providing a meaningful basis for clinical application of such a bioactive compound of herbal medicines.

Simultaneous determination of a variety of endocrine disrupting compounds in carrot, lettuce and amended soil by means of focused ultrasonic solid–liquid extraction and dispersive solid-phase extraction as simplified clean-up strategy

The present study is focused on the development of an analytical method based on focused ultrasonic solid–liquid extraction (FUSLE) followed by dispersive solid-phase extraction (dSPE) clean-up and liquid chromatography–triple quadrupole tandem mass spectrometry (LC–MS/MS) optimised for the simultaneous analysis of certain endocrine disrupting compounds (EDCs), including alkylphenols (APs), bisphenol A (BPA), triclosan (TCS) and several hormones and sterols in vegetables (lettuce and carrot) and amended soil samples. Different variables affecting the chromatographic separation, the electrospray ionisation and mass spectrometric detection were optimised in order to improve the sensitivity of the separation and detection steps. Under the optimised extraction conditions (sonication of 5min at 33% of power with pulse times on of 0.8s and pulse times off of 0.2s in 10mL of n-hexane:acetone (30:70, v:v) mixture using an ice bath), different dSPE clean-up sorbents, such as Florisil, Envi-Carb, primary-secondary amine bonded silica (PSA) and C18, or combinations of them were evaluated for FUSLE extracts before LC–MS/MS. Apparent recoveries and precision in terms of relative standard deviation (RSDs %) of the method were determined at two different fortification levels (according to the matrix and the analyte) and values in the 70–130% and 2–27% ranges, respectively, were obtained for most of the target analytes and matrices. Matrix-matched calibration approach and the use of labelled standards as surrogates were needed for the properly quantification of most analytes and matrices. Method detection limits (MDLs), estimated with fortified samples, in the ranges of 0.1–100ng/g for carrot, 0.2–152ng/g for lettuce and 0.9–31ng/g for amended soil were obtained. The developed methodology was applied to the analysis of 11 EDCs in both real vegetable bought in a local market and in compost (from a local wastewater treatment plant, WWTP) amended soil samples.

Simultaneous determination of perfluorinated compounds and their potential precursors in mussel tissue and fish muscle tissue and liver samples by liquid chromatography–electrospray-tandem mass spectrometry

An analytical method for the simultaneous determination in fish liver and muscle tissue and mussel samples of 14 perfluorinated compounds (PFCs), including three perfluoroalkylsulfonates (PFSAs), seven perfluorocarboxylic acids (PFCAs), three perfluorophosphonic acids (PFPAs) and perfluorooctanesulfonamide (PFOSA), and 10 potential precursors, including four polyfluoroalkyl phosphates (PAPs), four fluorotelomer saturated acids (FTCAs) and two fluorotelomer unsaturated acids (FTUCAs), was developed in the present work. Different clean-up strategies by means of solid-phase extraction (SPE) using a mix-mode weak anion exchanger (WAX), reverse phase Envi-Carb or a combination of them was optimized and evaluated for the clean-up of focused ultrasonic solid–liquid (FUSLE) extracts before the analysis by liquid chromatography–triple quadrupole tandem mass spectrometry (LC–MS/MS). Mix-mode WAX coupled in-line to Envi-Carb was finally selected since it rendered the cleanest extracts and minimum matrix effect. The FUSLE–SPE–LC–MS/MS methodology was validated in terms of recovery, precision and method detection limits (MDLs). Apparent recovery values in the 65–116%, 59–119% and 67–126% range and MDLs in the 0.1–2.7ng/g, 0.1–3.8ng/g and 0.2–3.1ng/g range were obtained for liver, mussel and fish muscle tissue samples, respectively. The method developed was applied to the analysis of grey mullet liver (Chelon labrosus) and mussel (Mytilus galloprovincialis) samples from the Basque Coast (North of Spain) and Yellowfin tuna muscle tissue (Thunnus albacares) samples from the Indian Ocean. To the best of our knowledge this is the first method that describes the simultaneous determination of 14 PFCs and 10 potential precursors in fish liver, fish muscle tissue and mussel samples. Besides, this is the first time that 8:2 monosubstituted polyfluorodecyl phosphate (8:2 monoPAP) and 8:2 disubstituted polyfluorodecyl phosphate (8:2 diPAP) were detected in mussel and tuna samples, respectively.

Simultaneous quantification of chrysophanol and physcion in rat plasma by ultra fast liquid chromatography-tandem mass spectrometry and application of the technique to comparative pharmacokinetic studies of Radix et Rhei Rhizoma extract alone and Dahuang Fuzi Decoction.

Most herbal medicines are prescribed in combination based on the theory of TCM to obtain synergistic effects or diminish the possible adverse reactions. Compatibility refers to the combination of two or more herbs based on the clinical settings and the properties of herbs. Chrysophanol and physcion are the main effective compounds in Radix et Rhizoma Rhei and Dahuang Fuzi Decoction which is the combination of Radix et Rhizoma Rhei, Radix Aconiti Lateralis Praeparata and Radix et Rhizoma Asari. However, chrysophanol and physcion are difficult to detect in vivo because of their low concentration and interference from endogenous compounds. The aim of this study is to develop a rapid, specific and sensitive ultra high performance liquid chromatography-triple quadrupole tandem mass method to simultaneously quantify chrysophanol and physcion in rat plasma, in order to better understand the pharmacokinetics and compatibility mechanism of Dahuang Fuzi Decoction for the first time. Multiple reaction monitoring (MRM) mode was applied for the quantitation at [M-H](-)m/z 253.0→m/z 225.1 for chrysophanol, [M-H](-) for m/z 283.1→m/z 240.0 physcion and [M-H](-)m/z 239.0→m/z 211.0 for IS. The analytes were separated on an Agilent Eclipse plus C18 column (100mm×2.1mm, 1.8μm) column within a total running time of 6.5min using a mixture of 3mM ammonium acetate in water and methanol (95:5, v/v) with a time program flow gradient according to the "plus gradient chromatography" theory. The inclusion of the ammonium acetate in the UFLC mobile phase dramatically improved the detection limit of the tested compounds and decreased the interference by matrix effects, which have been referred to as "LC-electrolyte effects". Finally, we demonstrated the application of a validated method in a comparative pharmacokinetic study of rats receiving an oral dose of Dahuang Fuzi Decoction or Radix et Rhei Rhizoma, the monarch drug in the prescription. Pharmacokinetic parameters showed significant difference between the two groups. The achieved comparative pharmacokinetic results were helpful to clarify the combination mechanism of Dahuang Fuzi Decoction.

Dihomo-isoprostanes—nonenzymatic metabolites of AdA—are higher in epileptic patients compared to healthy individuals by a new ultrahigh pressure liquid chromatography–triple quadrupole–tandem mass spectrometry method

Oxidative stress is a biochemical state in which reactive oxygen species are generated and it has been associated with pathological states including epilepsy. Therein, neuroprostanes (NeuroPs) and dihomo-isoprostanes (Dihomo-IsoPs)—a series of compounds formed nonenzymatically through free radical-induced DHA, n-6 DPA, and AdA peroxidation–are implicated in the pathophysiological status of various human neurological diseases. A new, robust, and selective analytical method for the determination of 10 NeuroPs/Dihomo-IsoPs in human urine, using solid-phase extraction and UHPLC–QqQ–MS/MS in the multiple reaction monitoring mode (using a negative electrospray ionization interface), was developed. Nine NeuroPs/Dihomo-IsoPs were identified in 15 epileptic patients, matched with healthy volunteers. Among them, 17-F2t-Dihomo-IsoP, Ent-7(R)-7-F2t-Dihomo-IsoP, and Ent-7-epi-7-F2t-Dihomo-IsoP, derived from adrenic acid (AdA), were significantly higher in epileptic patients than in healthy volunteers. The validated method provided a high-throughput assay with a limit of detection and limit of quantification for each analyte of 0.10–5.90ngmL−1 and 0.15–11.81ngmL−1, respectively. The intra- and interday variations were lower than 14%. Dihomo-IsoPs have been considered as potential markers of epilepsy for the first time and their measurement may increase the understanding of the role of oxidative stress in neurological diseases, in intra vitam studies. The present study highlights a potential role of Dihomo-IsoPs as biomarkers in persons with epilepsy, though its mechanisms and possible implications should be the subject of further investigations.

Determination of perfluorinated carboxylic acids in fish fillet by micro-solid phase extraction, followed by liquid chromatography–triple quadrupole mass spectrometry

In the current study, a simple, fast and efficient combination of protein precipitation and micro-solid phase extraction (μ-SPE) followed by liquid chromatography–triple quadrupole tandem mass spectrometry (LC–MS/MS) was developed for the determination of perfluorinated carboxylic acids (PFCAs) in fish fillet. Ten PFCAs with different hydrocarbon chain lengths (C5–C14) were analysed simultaneously using this method. Protein precipitation by acetonitrile and μ-SPE by surfactant-incorporated ordered mesoporous silica were applied to the extraction and concentration of the PFCAs as well as for removal of interferences. Determination of the PFCAs was carried out by LC–MS/MS in negative electrospray ionization mode. MS/MS parameters were optimized for multiple reaction monitoring of the analytes. 13C mass labelled PFOA as a stable-isotopic internal standard, was used for calibration. The detection limits of the method ranged from 0.97ng/g to 2.7ng/g, with a relative standard deviation of between 5.4 and 13.5. The recoveries were evaluated for each analyte and were ranged from 77% to 120%. The t-test at 95% confidence level showed that for all the analytes, the relative recoveries did not depend on their concentrations in the explored concentration range. The effect of the matrix on MS signals (suppression or enhancement) was also evaluated. Contamination at low levels was detected for some analytes in the fish samples. The protective role of the polypropylene membrane used in μ-SPE in the elimination of matrix effects was evaluated by parallel experiments in classical dispersive solid phase extraction. The results evidently showed that the polypropylene membrane was significantly effective in reducing matrix effects.

Using pollen grains as novel hydrophilic solid-phase extraction sorbents for the simultaneous determination of 16 plant growth regulators

In this article, pollen grains were for the first time used as a hydrophilic solid-phase extraction (HILIC-SPE) sorbent for the determination of 16 plant growth regulators (PGRs) in fruits and vegetables. Fourier transform infrared spectroscopy (FT-IR), scanning electronic microscopy (SEM) and nitrogen sorption porosimetry (NSP) were used to investigate the chemical structure and the surface properties of the pollen grains. Pollen grains exhibited an excellent adsorption capacity for some polar compounds due to their particular functional groups. Several parameters influencing extraction performance were investigated. A green and simple HILIC-SPE-method using pollen grain cartridge for purification of fruit and vegetable extractions, followed by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) was established. Good linear relationships were obtained for 16 PGRs with correlation coefficients (R) above 0.9980. The limits of detection (LODs) of 16 PGRs in cucumber were in the range of 0.01–1.10μg·kg−1. Reproducibility of the method was evaluated by intra-day and inter-day precisions with relative standard deviations (RSDs), which were less than 14.4%. We successfully applied this methodology to analyze 16 PGRs in 8 different kinds of fruits and vegetables. The recoveries from samples spiked with 16 PGRs were from 80.5% to 119.2%, with relative standard deviations less than 15.0%.

Targeted metabolomics in cultured cells and tissues by mass spectrometry: Method development and validation

Metabolomics is the identification and quantitation of small bio-molecules (metabolites) in biological samples under various environmental and genetic conditions. Mass spectrometry provides the unique opportunity for targeted identification and quantification of known metabolites by selective reaction monitoring (SRM). However, reproducibility of this approach depends on careful consideration of sample preparation, chemical classes, and stability of metabolites to be evaluated. Herein, we introduce and validate a targeted metabolite profiling workflow for cultured cells and tissues by liquid chromatography–triple quadrupole tandem mass spectrometry. The method requires a one-step extraction of water-soluble metabolites and targeted analysis of central metabolites that include glycolysis, amino acids, nucleotides, citric acid cycle, and the hexosamine biosynthetic pathway. The sensitivity, reproducibility and molecular stability of each targeted metabolite were assessed under experimental conditions. Quantitation of metabolites by peak area ratio was linear with a dilution over a 4 fold dynamic range with minimal deviation R2=0.98. Inter- and intra-day precision with cells and tissues had an average coefficient of variation <15% for cultured cell lines, and somewhat higher for mouse liver tissues. The method applied in triplicate measurements readily distinguished immortalized cells from malignant cells, as well as mouse littermates based on their hepatic metabolic profiles.