Liquid Chromatography-electrospray Tandem Mass Research Articles

Sustainable chromatographic quantitation of multi-antihypertensive medications: application on diverse combinations containing hydrochlorothiazide along with LC–MS/MS profiling of potential impurities: greenness and whiteness evaluation

Cardiovascular disorders are among the leading causes of death worldwide, especially hypertension, a silent killer syndrome requiring multiple drug therapy for appropriate management. Hydrochlorothiazide is an extensively utilized thiazide diuretic that combines with several antihypertensive drugs for effective treatment of hypertension. In this study, sustainable, innovative and accurate high performance liquid chromatographic methods with diode array and tandem mass detectors (HPLC–DAD and LC–MS/MS) were developed, optimized and validated for the concurrent determination of Hydrochlorothiazide (HCT) along with five antihypertensive drugs, namely; Valsartan (VAL), Amlodipine besylate (AML), Atenolol (ATN), Amiloride hydrochloride (AMI), and Candesartan cilextil (CAN) in their diverse pharmaceutical dosage forms and in the presence of Chlorothiazide (CT) and Salamide (DSA) as HCT officially identified impurities. The HPLC–DAD separation was achieved utilizing Inertsil ODS-3 C18 column (250 × 4.6 mm, 5 μm) attached with photodiode array detection at 225.0 nm. Gradient elution was performed utilizing a mixture of solvent A (20.0 mM potassium dihydrogen phosphate, pH 3.0 ± 0.2, adjusted with phosphoric acid) and solvent B (acetonitrile) at ambient temperature. Linearity ranges were 0.1–100.0 µg/mL for HCT, VAL, AML and CAN, 0.05 –100.0 µg/mL for both ATN and AMI and 0.05–8.0 µg/mL for both CT and DSA. Additionally, this work describes the use of liquid chromatography–electrospray–tandem mass spectrometry for the accurate detection and quantification of the impurities; CT and DSA in the negative mode utilizing triple quadrupole mass spectrometry. The linearity ranges for those impurities were 1.0–200.0 ng/mL and 5.0–200.0 ng/mL for CT and DSA, respectively. Developed methods’ validation was achieved in accordance with International Conference on Harmonization (ICH) guidelines. Upon applying liquid chromatographic techniques for the drug analysis, a green and sustainable assessment have to be handled due to the consumption of energy and many solvents. Through the use of the HEXAGON, Analytical Greenness (AGREE) and White Analytical Chemistry (WAC) tools, greenness and sustainability have been statistically assessed. The optimized HPLC–DAD and LC–MS/MS methods were fast, accurate, precise, and sensitive, and consequently could be applied for conventional analysis and quality control of the proposed drugs in their miscellaneous dosage forms for the purpose of reducing laboratory wastes, time of the analysis time, effort, and cost.Graphical

Simultaneous pharmacokinetic assessment of phytopharmaceuticals in fenugreek extract using LC-MS/MS in Sprague-Dawley rats.

Fenugreek seeds are used in numerous marketed herbal formulations with therapeutic benefits. Some of its bioactive components such as 4-hydroxyisoleucine, trigonelline, raffinose, and pinitol are reported to possess potential therapeutic activities, such as antibacterial, antidiabetic, stomach stimulant, and anti-invasive, against hyperandrogenism and other allied diseases, including polycystic ovary syndrome. A fully validated, selective, and sensitive bioanalytical method for the simultaneous rapid quantification of the aforementioned bioactive components has been developed using hyphenated liquid chromatography electrospray tandem mass spectrometry. The analytes were separated within 5min using gradient elution in a C18 column at a flow rate of 0.5ml/min. Plasma protein precipitation technique was employed to isolate the analytes from the samples. Oral pharmacokinetic profile of the four bioactive components in Sprague-Dawley rats was further evaluated using noncompartmental analysis using Phoenix WinNonlin software.

Saffron Characterization by a Multidisciplinary Approach

Saffron is a spice obtained from the drying process of the stigmas of the flower Crocus sativus Linnaeus. It is well known that the organoleptic characteristics of this spice are closely linked to the production area and harvesting year. The present work aims to evaluate whether saffron samples produced in different years and origins present sensibly different crocin profiles. To achieve this goal, 120 saffron samples were harvested between 2016 and 2020 in four different Italian areas. The crocins were analysed, identified, and quantified by high-performance liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) in multiple reaction monitoring mode (MRM). Subsequently, ANOVA-simultaneous component analysis (ASCA) was used to evaluate whether the origin and annuity significantly affected the composition of the crocins. ASCA confirmed the relevance of these effects. Eventually, soft independent modelling by class analogy (SIMCA) models were created for each of the four different origins. Mixtures of saffron from different areas were also prepared to test the robustness of the models. SIMCA provided satisfying results; in fact, models provided 100% sensitivity for three origins (Cascia, Sardinia, and Città della Pieve) on the external test set (48 samples) and 88% (sensitivity on the external test set) for the Spoleto class.

Direct acetylation for full analysis of polysaccharides in edible plants and fungi using reverse phase liquid chromatography-multiple reaction monitoring mass spectrometry

It is vitally important to characterize polysaccharides by monosaccharide composition method. In this study, a direct acetylation strategy combined with reversed-phase liquid chromatography electrospray tandem multiple reaction monitoring mass spectrometry (RPLC-ESI-MRM-MS) was developed for simultaneous determination of 8 aldoses (Glc, Gal, Man, Ara, Xyl, Rib, Rha and Fuc), a ketose (Fru), 2 alditols (Glc-ol and Man-ol) and 2 uronic acids (GlcA and GalA) on a high-pressure resistant reversed-phase column. Employing 1-MeIm as catalyst for direct acetylation, even though no DMSO was used to inhibit the transformation of configurations, each carbohydrate still produced a single chromatographic peak in RPLC conditions due to the ɑ- and β- isomers merged together. Except for Fru and Man, all the other 11 carbohydrates were base-line separated in a 1.7 µm CYANO column. Therefore, correction factor method is further proposed to perfectly solve co-elution problem of Fru and Man because of occurrence of a specific Q3 ion for aldoses rather than ketose. The result was verified on a 1.7 µm Fluoro-Phenyl column with a full separation of Fru and Man. Herein, the established direct acetylation as followed RPLC-ESI-MRM-MS method was successfully applied for compositional analysis of complex polysaccharides from edible plants and fungi.

Occurrence and human health risk assessment of antibiotics in cultured fish from 19 provinces in China.

The occurrence of antibiotics and potential health risk of 300 cultured fish samples from 19 provinces in China were investigated. The levels of 28 antibiotics (15 fluoroquinolones, 4 tetracyclines, 8 macrolides and rifampin) in 8 fish species were measured through liquid chromatography electrospray tandem mass spectrometry. As a result, 10 antibiotics were detected with an overall detection frequency of 24.3%, and the individual detection frequency of antibiotics ranged from 0.33 to 16.7%. The extremely high concentrations (above 100 µg/kg) of doxycycline and erythromycin were found in the samples. Antibiotics with high detection frequency was noticed in largemouth bass (41.2%), followed by snakehead (34.4%) and bream (31.2%). Specifically, Heilongjiang, Xinjiang, Qinghai and Gansu presented high detection frequency values of more than 60%. Moreover, the highest mean concentration was observed in Shandong, and the concentration covered from 34.8 µg/kg to 410 µg/kg. Despite the high detection frequency and levels of antibiotics were found in samples, ingestion of cultured fish was not significantly related to human health risks in China, according to the calculated estimated daily intakes and hazard quotients. These results provided us the actual levels of antibiotics in cultured fish and human health risk assessment of consuming fishery products.

Quantitative proteomics reveals protein dysregulation during T cell activation in multiple sclerosis patients compared to healthy controls

BackgroundMultiple sclerosis (MS) is an autoimmune, neurodegenerative disorder with a strong genetic component that acts in a complex interaction with environmental factors for disease development. CD4+ T cells are pivotal players in MS pathogenesis, where peripherally activated T cells migrate to the central nervous system leading to demyelination and axonal degeneration. Through a proteomic approach, we aim at identifying dysregulated pathways in activated T cells from MS patients as compared to healthy controls.MethodsCD4+ T cells were purified from peripheral blood from MS patients and healthy controls by magnetic separation. Cells were left unstimulated or stimulated in vitro through the TCR and costimulatory CD28 receptor for 24 h prior to sampling. Electrospray liquid chromatography-tandem mass spectrometry was used to measure protein abundances.ResultsUpon T cell activation the abundance of 1801 proteins was changed. Among these proteins, we observed an enrichment of proteins expressed by MS-susceptibility genes. When comparing protein abundances in T cell samples from healthy controls and MS patients, 18 and 33 proteins were differentially expressed in unstimulated and stimulated CD4+ T cells, respectively. Moreover, 353 and 304 proteins were identified as proteins exclusively induced upon T cell activation in healthy controls and MS patients, respectively and dysregulation of the Nur77 pathway was observed only in samples from MS patients.ConclusionsOur study highlights the importance of CD4+ T cell activation for MS, as proteins that change in abundance upon T cell activation are enriched for proteins encoded by MS susceptibility genes. The results provide evidence for proteomic disturbances in T cell activation in MS, and pinpoint to dysregulation of the Nur77 pathway, a biological pathway known to limit aberrant effector T cell responses.

Phytotoxic effect and molecular mechanism induced by graphene towards alfalfa (Medicago sativa L.) by integrating transcriptomic and metabolomics analysis

Although the widespread use of nanoparticles has been reported in various fields, the toxic mechanisms of molecular regulation involved in the alfalfa treated by nanomaterials is still in the preliminary research stage. In this study, Bara 310 SC (Bara, tolerant genotype) and Gold Empress (Gold, susceptible genotype) were used to investigate how the leaves of alfalfa interpret the physiological responses to graphene stress based on metabolome and transcriptome characterizations. Herein, graphene at different concentrations (0, 1% and 2%, w/w) were selected as the analytes. Physiological results showed antioxidant defence system and photosynthesis was significantly disturbed under high environmental concentration of graphene. With Ultra high performance liquid chromatography electrospray tandem mass spectrometry (UPLC-ESI-MS/MS), 406 metabolites were detected and 62/13 and 110/58 metabolites significantly changed in the leaves of Gold/Bara under the 1% and 2%-graphene treatments (w/w), respectively. The most important metabolites which were accumulated under graphene stress includes amino acids, flavonoids, organic acids and sugars. Transcriptomic analysis reveals 1125 of core graphene-responsive genes in alfalfa that was robustly differently expressed in both genotypes. And differential expression genes (DEGs) potentially related to photosynthetic enzymes, antioxidant enzymes, amino acids metabolism, and sucrose and starch metabolic which finding was supported by the metabolome study. Gold was more disturbed by graphene stress at both transcriptional and metabolic levels, since more stress-responsive genes/metabolites were identified in Gold. A comprehensive analysis of transcriptomic and metabolomic data highlights the important role of amino acid metabolism and nicotinate and nicotinamide metabolism pathways for graphene tolerance in alfalfa. Our study provide necessary information for better understanding the phytotoxicity molecular mechanism underlying nanomaterials tolerance of plant.

Determination and Occurrence of Mineralocorticoids in Taihu Lake of China

We developed a sensitive method for monitoring six natural (aldosterone) and synthetic mineralocorticoids (canrenone, spironolactone, 7β-spironolactone, 7α-thio spironolactone, and 7α-thiomethyl spironolactone) in sediment and water using ultra-performance liquid chromatography–electrospray tandem mass spectrometry, and then 30 water and 30 sediment samples were analyzed to reveal their occurrence and distributions in Taihu Lake. All target six mineralocorticoids were detected in sediment and water samples with the detection frequencies as high as 96–100%. The median concentrations of mineralocorticoids ranged from 0.04 ng/L (7α-thiomethyl spironolactone) to 14 ng/L (aldosterone) in water and 0.01 ng/g (7β-spironolactone and canrenone) to 1.44 ng/g (aldosterone) in sediment in dry weight. Natural aldosterone was the predominant mineralocorticoid detected in both water and sediment samples, indicating the mineralocorticoid pollution in Taihu Lake was mainly derived from human and/or animal excrement rather than pharmaceutical industry and usage. Two metabolites 7β-spironolactone and 7α-thio spironolactone were first found in this study. Low ratios of metabolites to spironolactone were observed in sediment (0.05–0.75) in contrast to water (0.12–2.26), indicating that spironolactone was prone to degradation in water phase compared to sediment environment.

Quantitative Proteomics Reveals Protein Dysregulation During T Cell Activation in Multiple Sclerosis Patients

Multiple sclerosis (MS) is an autoimmune, neurodegenerative disorder with a strong genetic component that acts in a complex interaction with environmental factors for disease development. CD4+ T cells are pivotal players in MS pathogenesis, where peripherally activated T cells migrate to the central nervous system leading to demyelination and axonal degeneration. In the current study, we evaluated dysregulation of CD4+ T cell activation at the protein level using electrospray liquid chromatography-tandem mass spectrometry to get insights into immune-cell processes in MS. Our study highlights the importance of CD4+ T cell activation for MS, as proteins that change in abundance upon T cell activation are enriched for proteins encoded by MS susceptibility genes. Our results provide evidence for proteomic disturbances in T cell activation in MS, and pinpoint to dysregulation of the Nur77 pathway, a biological pathway known to limit aberrant effector T cell responses.

Exposure to antibiotics and mental disorders in children: a community-based cross-sectional study.

Although exposure to antibiotics at a critical developmental time window has been implicated in mental health in observational and experimental studies, very limited bio-monitoring data are available for exposure to antibiotics associated with child mental disorders. The goal of our study was to examine the association between urinary exposure of children to antibiotics and mental health. The participants were 278 children from 256 eligible families in the urban-rural fringe of Fuyang city in China since June in 2017. A single-point urine sample was collected to measure the antibiotic concentrations to characterize the exposure levels. A total of 45 antibiotics from nine classes and their two metabolites were monitored through liquid chromatography electrospray tandem mass spectrometry. We used multivariable regressions to estimate the covariate-adjusted associations between urine-antibiotic concentrations and mental impairments, as assessed using the parent version of Strengths and Difficulties Questionnaire. Among the participants, ciprofloxacin was associated with an increased risk of mental disorders at both lower concentrations (OR = 4.06; 95% CI 1.69-9.78) and higher concentrations OR = 6.04; 95% CI 2.59-14.08). After categorizing the detected antibiotics, the positive associations were observed between abnormal score in total difficulties and higher levels exposure to fluoroquinolones (OR = 2.83, 95% CI 1.38-5.80) and antibiotics preferred for veterinary use (PVAs) (OR = 3.20; 95% CI 1.41-7.27), respectively. Our findings suggest that ciprofloxacin, fluoroquinolones and PVAs, probably from contaminated food or environment, may be associated with child mental disorders.

Simultaneous analysis and risk assessment of Quizalofop, Acifluorfen, bentazone and its metabolites residues in peanut and straw under field conditions of China

In this study, we developed and validated a simple and robust method for simultaneous determination of quizalofop-p-ethyl, acifluorfen, bentazone and its two metabolites residues in peanut and straw. All samples were extracted by 0.8% formic acetonitrile (v:v) and pure water. The peanut samples were cleaned up by florisil, while the straw samples were cleansed using florisil and graphitized carbon black (GCB) by adding a desorption procedure at 55 °C. The residues were determined by a high performance liquid chromatography electro-spray ionization tandem mass spectrometry (HPLC-MS/MS). The mean recoveries of five analytes in both matrixes ranged from 77% to 110% with relative standard deviations (RSDs) <12.6%. The limits of quantification (LOQs) were 0.01 mg/kg in peanut and 0.02 mg/kg in straw. And the limits of detection (LODs) ranged from 0.047 to 0.356 μg/L. The established method was successfully applied to determine the terminal residues of the five analytes in peanut and straw samples from supervised field trials. The results showed that the residues of all analytes were less than LOQs in peanut while that was up to 0.031 mg/kg in straw. Based on residues data in peanut, chronic dietary intake risk of three pesticides was evaluated, and the results showed that the risk of all pesticides were negligible for Chinese residents.

Antibiotic exposure and potential risk of depression in the Chinese elderly: a biomonitoring-based population study.

To examine the associations between urinary antibiotics from various sources and depression in the elderly using the biomonitoring method. In the current study, we investigated 990 elderly individuals (≥ 60 years old) from a community-based elderly cohort in West Anhui, China. The participants were interviewed by the Geriatric Depression Scale and self-developed questionnaires. A total of 45 antibiotics belonging to nine categories were screened in urine samples by the developed liquid chromatography electrospray tandem mass spectrometry method. Creatinine-corrected concentrations of antibiotics in urines were used to assess their exposure. Logistic regression analysis was employed to test the relationships between exposure to antibiotics and depression. Compared to the control group, the multinomial logistic regression analyses showed the elderly exposed to higher concentrations of azithromycin (OR = 1.81, 95% CI: 1.09-3.00) and sulfaclozine (OR = 1.54, 95% CI: 1.05-2.28) had increased risks of depression, respectively. After categorizing the detected antibiotics, tetracyclines (OR = 1.48, 95% CI: 1.02-2.16) and veterinary antibiotics (VAs) (OR = 1.53, 95% CI: 1.06-2.20) were positively correlated with increased risks of depression. After stratified by sex, the VAs (OR = 2.04, 95% CI: 1.13-3.71) at higher concentrations were associated with elevated risks of depression in males, while the associations between depression and antibiotic exposures were observed in tetracyclines (OR = 1.74, 95% CI: 1.04-2.85) and all antibiotics (OR = 2.24, 95% CI: 1.01-2.94) at higher levels in females, respectively. Notably, after the stratification by age, the significant associations were mainly present in the subjects under the age of 70. Our findings reveal that azithromycin, sulfaclozine, tetracyclines, and the VAs were significantly associated with elevated risks of depression in the elderly. Importantly, sex- and age-specific differences were observed in the associations between antibiotic exposures and depression.

Abstract PO-006: Tracking S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH) biosynthesis pathway using stable isotope-resolved metabolomics (SIRM)

Abstract S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH) are low abundance, key metabolites in one-carbon metabolism. The ratio of SAM/SAH controls epigenetic DNA and histone methylation, which is central to regulating gene expression in both normal and disease states including cancers (1,2). Stable isotope resolved metabolomics (SIRM) is a powerful approach for mapping metabolic networks in cells and tissues, which employs untargeted ultrahigh resolution MS1 to quantify a full set of isotopologues for a large number of metabolites (3). This demand makes it difficult to implement liquid chromatography (LC)-based analysis. We have developed a simple, rapid and robust LC-free method for analyzing SAM and SAH in mouse tissue, A549 cells, and human plasma. This method has sub nmol detection and is fully compatible with SIRM analysis of many other metabolites. Flash frozen mouse liver tissues or A549 cells were quenched and extracted with ice-cold acetonitrile:water:chloroform (2:0.75:1, v/v). Prior to extraction, A549 cells were grown for 24 h in the presence of either 13C6-glucose or 13CH3-methionine. No extraction was applied to human plasmas. SAM and SAH were captured by solid phase extraction (SPE) using a Bond-Elut phenylboronic acid (PBA) column (Agilent). SAM and SAH levels in liver tissues and plasma obtained by our method was comparable to those found by other methods (4). Tracing with 13C6 glucose showed extensive incorporation of label into the ribose ring and purine of the adenosyl subunit in both SAM and SAH. In contrast, tracing with 13CH3-methioinine showed predominant, single carbon label in SAM and none in SAH, suggesting the prevalence in the incorporation of exogenous methionine into SAM and turnover into SAH. Thus, it is practical to determine SAM and SAH turnover in the methionine cycle in various biological samples, which provides important insights into epigenetic metabolism. Funding: This work was supported in part by grants: 1R01ES022191-01 (to TWMF and RMH), 1P01CA163223-01A1 (to ANL and TWMF), and 1U24DK097215-01A1 (to RMH, TWMF, and ANL).

Variation of the Main Alkaloid Content in Equisetum palustre L. in the Light of Its Ontogeny.

Marsh horsetail (Equisetum palustre L.) is one of the most poisonous plants of wet grasslands in the northern hemisphere, which poses a major health threat to livestock. Available data on the levels of its main alkaloids are currently contradictory due to the inadequate analytical methods and the wide variation in toxicity levels reported. Here, we tested the hypothesis that the ontogenetic stage of plant development may explain a significant part of the variations in the main Equisetum-type alkaloids. Two populations of marsh horsetail were sampled over two growing seasons. The plant material was classified according to their developmental stages and subsequently the main alkaloids were determined by hydrophilic interaction liquid chromatography and high-performance liquid chromatography electrospray tandem mass spectrometry (HILIC HPLC-ESI-MS/MS) analysis. ANOVA revealed significant effects of the ontogenetic stage but not the site on the main Equisetum-type alkaloids (sum of palustrine and palustridiene) ranging from 213 to 994 mg/kg dry matter (DM). The highest alkaloid content was found in the stages of early development. Not the season itself, but the growth temperature co-influenced the alkaloid content. Our results help to resolve the seemingly contradictory information provided by previous studies on the toxicity of E. palustre and are of practical relevance for the prevention of contamination risks in wet grassland use.

An essay on ecosystem availability of Nicotiana glauca graham alkaloids: the honeybees case study

BackgroundInvasive plant species pose a significant threat for fragile isolated ecosystems, occupying space, and consuming scarce local resources. Recently though, an additional adverse effect was recognized in the form of its secondary metabolites entering the food chain. The present study is elaborating on this subject with a specific focus on the Nicotiana glauca Graham (Solanaceae) alkaloids and their occurrence and food chain penetrability in Mediterranean ecosystems. For this purpose, a targeted liquid chromatography electrospray tandem mass spectrometric (LC–ESI–MS/MS) analytical method, encompassing six alkaloids and one coumarin derivative, utilizing hydrophilic interaction chromatography (HILIC) was developed and validated.ResultsThe method exhibited satisfactory recoveries, for all analytes, ranging from 75 to 93%, and acceptable repeatability and reproducibility. Four compounds (anabasine, anatabine, nornicotine, and scopoletin) were identified and quantified in 3 N. glauca flowers extracts, establishing them as potential sources of alien bio-molecules. The most abundant constituent was anabasine, determined at 3900 μg/g in the methanolic extract. These extracts were utilized as feeding treatments on Apis mellifera honeybees, resulting in mild toxicity documented by 16–18% mortality. A slightly increased effect was elicited by the methanolic extract containing anabasine at 20 μg/mL, where mortality approached 25%. Dead bees were screened for residues of the N. glauca flower extracts compounds and a significant mean concentration of anabasine was evidenced in both 10 and 20 μg/mL treatments, ranging from 51 to 92 ng/g per bee body weight. Scopoletin was also detected in trace amounts.ConclusionsThe mild toxicity of the extracts in conjunction with the alkaloid and coumarin residual detection in bees, suggest that these alien bio-molecules are transferred within the food chain, suggesting a chemical invasion phenomenon, never reported before.

Metabolomic and transcriptomic analyses reveal the regulation of pigmentation in the purple variety of Dendrobium officinale

We performed an integrated analysis of the transcriptome and metabolome from purple (Pr) and normal cultivated varieties (CK) of Dendrobium officinale to gain insights into the regulatory networks associated with phenylpropanoid metabolism and to identify the key regulatory genes of pigmentation. Metabolite and transcript profiling were conducted by ultra-performance liquid chromatography electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) and RNA sequencing. Pr had more flavonoids in the stem than did CK. Metabolome analyses showed that 148 differential metabolites are involved in the biosynthesis of phenylpropanoids, amino acids, purines, and organic acids. Among them, the delphinidin and quercetin derivatives were significantly higher in Pr. A total of 4927 differentially expressed genes (DEGs) were significantly enriched (p ≤ 0.01) in 50 Gene Ontology (GO) terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed significantly enriched phenylpropanoid biosynthesis and phytohormone signal transduction in Pr versus CK. The expression levels of flavanone 3-hydroxylase (F3H) and leucoanthocyanidin dioxygenase (LDOX) affected the flux of dihydroflavonol, which led to a color change in Pr. Moreover, DEG enrichment and metabolite analyses reflected flavonoid accumulation in Pr related to brassinosteroid (BR) and auxin metabolism. The results of this study elucidate phenylpropanoid biosynthesis in D. officinale.

Determination of Amphetamine and Methamphetamine in Human Hair by Gas Chromatography Mass Spectrometry

This article develops a combined solid phase extraction (SPE) and gas chromatography – mass spectrometry (GC-MS) procedure for determining amphetamine-type stimulants Amphetamine (AM) and Methamphetamine (MA) in human hair. Hair samples were incubated in methanol containing 1% hydrochloric acid in 18 hours and then subjected to SPE. The obtained extracts were evaporated to dryness, derivatized with heptafluorobutyric anhydride (HFBA) at 70 °C for 30 minutes prior to GC–MS analysis. Gas chromatography mass spectrometry was run on HP5-MS column (30 m × 0.25 mm × 0.25 µm) with detector MS 5975C. Experimentally, the proposed method proved sensitive, simple and time-saving, but quite accurate with a low limit of detection (LOD = 0.05ng/mg) and quantitation (LOQ = 0.15ng/mg).&#x0D; Keywords:&#x0D; SPE, GC – MS, hair samples, amphetamine, methamphetamine.&#x0D; References&#x0D; [1] Ming-Ren Fuh, Ti-Yu Wu and Tzuen-Yeuan Lin, Determination of amphetamine and methamphetamine in urine by solid phase extraction and ion-pair liquid chromatography–electrospray–tandem mass spectrometry Talanta, 68 (3) (2006), 987-991. https://doi.org/10.1016/j.talanta.2005.06.057[2] Naresh C. Jain, Thomas C. Sneath, and Robert D. Budd, Rapid Gas-Chomatographic Determination of Amphetamine and Methamphetamine in urine, Clinical Chemistry, 20 (11) (1974) 1460-1462. https://doi.org/10.1093/clinchem/20.11.1460.[3] Dong-liang Lin, Rea-Ming Yin, Ray H. Liu, Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Amphetamine, Methamphetamine, 3,4-Methylenedioxy- amphetamine and 3,4-Methylenedioxymethamphetamine in Human Hair and Hair Sections, Journal of Food and Drug Analysis, 13(2) (2005) 193-200. https://doi.org/10.38212/2224-6614.2526[4] María Jesús Tabernero, Maria Linda Felli, Ana María Bermejo, Marcello Chiarotti, Determination of ketamine and amphetamines in hair by LC/MS/MS, Anal Bioanal Chem, 395(2009), 2547–2557. https://doi.org/10.1007/s00216-009-3163-4.[5] D.V. Doan, D.Q. Huy, N.D. Hue, T.M. Tri, Determination of methamphetamine in urine samples by gas chromatography mass spectrometry combined with solid phase extraction technique, Journal of Science and Technology 47 (6) (2009) 53-58 (in Vietnamese).[6] Rodger L. Foltz, Allison F. Fentiman, Ruth B. Foltz, GC/MS Assays for Abused Drugs in Body Fluids, National Institute on Drug Abuse, Maryland, 1980.[7] AOAC, Appendix F: Guidelines for Standard Method Performance Requirements, AOAC official methods of analysis, Maryland, 2016.[8] Eunyoung Han, Martin P. Paulus, Marc Wittmann, Heesun Chung, Joon myong Song, Hair analysis and self-report of methamphetamine use by methamphetamine dependent individuals, Journal of Chromatography B, 879 (2011) 541–547. https://doi.org/10.1016/j.jchromb.2011.01.002.&#x0D; &#x0D; &#x0D;

Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

A sensitive, reproducible reverse-phased high performance liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method with simple sample preparation was developed for the simultaneous determination of a wide range of ceramides, diacylglycerols (DAGs) in cultured cells. Chromatographic separation of the compounds was achieved in a 14-minute run using a C8 column with a gradient elution by methanol and 10 mM ammonium acetate buffer as mobile phase at a flow rate of 0.5 ml/min. Various ceramides, DAGs were detected with a triple quadrupol system in multiple reaction monitoring mode, which is based on a soft positive electrospray ionization. The usual sample preparation process was shortened by the application of pure methanol for the extraction instead of the widely used methanol/chloroform mixture. C17:0 ceramide which does not occur in the cell samples, was used as an internal standard. The sample preparation process was optimized and the methodology was tested on a human hepatocarcinoma cell culture. Our results clearly showed accumulation of some ceramides and DAGs in the cells treated with BSA-conjugated palmitate for 8 hours. Since both ceramides and DAGs are important lipid intermediates and signal messengers, alteration in their cellular levels have major impact on cell functions, and thus our novel analytic method can be widely used in lipotoxicity research. The presented technique can be further developed to measure other intermediates of ceramide synthesis and other derivatives of DAGs as well.

Bioactive compounds profile, enzyme inhibitory and antioxidant activities of water extracts from five selected medicinal plants

Medicinal plants are a potentially renewable source of biomolecules. The aim of the current study was to investigate the bioactive compounds, the antioxidant capacity and the enzyme inhibitory effects of aqueous extracts from Camellia sinensis L., Erica arborea L., Ilex paraguariensis A. St. Hilaire, Rosmarinus officinalis L., and Thymus vulgaris L. The extraction yields were between 8.54 and 32.62 %. The contents of total phenolic compounds varied from 31.55 to 199.06 mg GAEs/g extract. Thirteen bioactive compounds were identified using liquid chromatography–electrospray tandem mass spectrometry and fifteen compounds were detected in the five species (Protocatechuic acid, 3,4-dihydroxyphenylacetic acid, (+)-catechin, chlorogenic acid, 4-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, 3-hydroxybenzoic acid, verbascoside, p-coumaric acid, ferulic acid, hesperidin, hyperoside, and rosmarinic acid). Ilex paraguariensis and R. officinalis showed the highest α-amylase and α-glucosidase inhibitory activities. The correlation analysis showed that caffeic acid and pinoresinol were significantly correlated with α-amylase and α- glucosidase inhibitory activities, respectively. Principal component analysis and cluster analysis grouped the studied species into three main clusters: the first group included E. arborea, the second group included I. paraguariensis, and the third group included C. sinensis, R. officinalis, and T. vulgaris.

Plantago lanceolata as a source of health-beneficial phytochemicals: Phenolics profile and antioxidant capacity

Plantain (Plantago) species are traditionally used as food products and folkloric medicines in many countries. This study evaluated the antioxidant effects, phytochemical content, and phenolics profile of Plantago lanceolata (ribwort plantain) as an important herb in Turkey. Five different methods were used to measure the antioxidant activity including β-carotene bleaching, DPPH radical scavenging, reducing power, metal chelating, and phosphomolybdenum total antioxidant assays. Total content of phenolics and flavonoids were also determined. Profiling of phenolic compounds was done using liquid chromatography–electrospray tandem mass spectrometry. Methanolic extracts showed the highest antiradical and reducing activities. Water extracts had strong β-carotene bleaching (71% at 0.4 mg/mL) and ferrous ion chelating (95% at 0.25 mg/mL) abilities. Methanol extracts had high antioxidant capacity in the phosphomolybdenum assay (145 mg AAE/g extract). Phenolics profiling showed 28 phenolic acids, flavonoid, and phenylethanoid glycoside compounds. A high amount of verbascoside (94.8 mg/g dry aerial parts of plant) was obtained followed by chlorogenic acid, rosmarinic acid, hesperidin, and hyperoside. Plantago lanceolata has considerable potential for use in new food and pharmaceutical products. Also, it could be regarded as a rich source of verbascoside.