Ultra-fast Liquid Chromatography Mass Spectrometry Research Articles

HPLC/Fluorescence-Diode Array Detection for Rapid and Reliable Determination of Illegal Synthetic Drugs in Male Sexual Herbal and Honey Remedies: Comparative Study with UFLC-MS.

Nutraceuticals (NTCs), as honey and tablets with herbal extract are subjected to adulteration. For NTCs claimed to enhance sexual performance, synthetic drugs (sildenafil, tadalafil, avanafil, vardenafil, and dapoxetine) are common adulterants, so they were selected to be simultaneously analyzed in the current study. Natural aphrodisiacs (icariin and yohimbine) are claimed to be present in many fake NTCs, so they were also included in the study. In order to achieve the target of the current study, three liquid chromatographic methods with different unique detectors were developed and validated. High performance liquid chromatography (HPLC) with fluorescence detection enables rapid and reliable determination of natively fluorescent yohimbine, tadalafil vardenafil, and dapoxetine and it is the first report to analyze these compounds as adulterants in counterfeit NTC. Although the diode-array detector (DAD) enables the analysis of the seven adulterants, the fluorescence detector (FLD) shows better sensitivity and selectivity with lower LOQs and LODs. On the other hand, ultra-fast liquid chromatography-mass spectrometry (UFLC-MS) offers the advantages of peak identity confirmation, and it is of comparable sensitivity and selectivity to HPLC-FLD. One or more of these synthetic drugs were found in the analyzed NTCs while natural aphrodisiacs were absent. Aphrodisiac nutraceuticals, NTCs, were analyzed for adulterants: five aphrodisiac synthetic drugs (adulterants) and two natural claimed aphrodisiacs. UFLC-MS and HPLC-DAD/FLD were compared for illicit NTCs analysis; all NTCs show the presence of synthetic aphrodisiacs and the absence of natural ones.

Comparative Analysis of Free Amino Acids and Nucleosides in Different Varieties of Mume Fructus Based on Simultaneous Determination and Multivariate Statistical Analyses.

Mume Fructus (MF) contains a variety of organic acids, free amino acids, and nucleoside components, and studies have not yet analyzed the relationship between the components of free amino acids and nucleosides with the varieties of MF. A rapid and sensitive method was established for simultaneous determination of 21 free amino acids and 9 nucleosides in MF by ultrafast liquid chromatography-mass spectrometry. The analysis was carried out on a Waters XBridge Amide column (100 mm × 2.1 mm, 3.5 μm) with elution by the mobile phase of 0.2% aqueous formic acid (A) and 0.2% formic acid acetonitrile (B) at a flow rate of 0.2 mL/min with 1 μL per injection. The column temperature was maintained at 30°C. The target compounds were analyzed by the positive ion multiple reaction monitoring (MRM) mode. The comprehensive evaluation of the samples was carried out by principal component analysis (PCA) and technique for order preference by similarity to an ideal solution (TOPSIS) analysis. Results showed the method could simultaneously determine 30 components in MF. The content of total analytes in six mainstream varieties was different, exhibited the order Nangao > Daqingmei > Zhaoshuimei > Yanmei > Shishengme > Baimei, and aspartic acid and adenosine were the most abundant amino acid and nucleoside. PCA and OPLS-DA could easily distinguish the samples, and 11 components could be chemical markers of sample classification. TOPSIS implied that the quality of Nangao and Daqingmei was superior to the other varieties. The results could provide a reliable basis for quality evaluation and utilisation of medicinal and edible MF.

Lnc00462717 regulates the permeability of the blood-brain tumor barrier through interaction with PTBP1 to inhibit the miR-186-5p/Occludin signaling pathway.

Blood-brain tumor barrier (BTB) severely restricts the efficient delivery of chemotherapeutic drugs into brain tumor tissue, which is a critical obstacle for glioma treatment. Recently, long noncoding RNAs (lncRNAs) have shown as regulation factors of numerous biological processes. In this study, we identified that Lnc00462717 was upregulated in glioma endothelial cells (GECs), and that knockdown of Lnc00462717 significantly increased the BTB permeability. Both bioinformatics and RNA immunoprecipitation (RIP) results revealed that Lnc00462717 interacts with polypyrimidine tract binding protein (PTBP1). Moreover, overexpression of PTBP1 significantly reversed the increase in BTB permeability caused by siLnc00462717. Furthermore, the binding sites between miR-186 and PTBP1 as well as between miR-186 and 3'UTR of Occludin mRNA were confirmed by RIP and luciferase assays, respectively. And the interaction of Lnc00462717 and PTBP1 significantly facilitated the binding of PTBP1 to 3'UTR of Occludin mRNA and then blocked the miR-186-5p-induced downregulation of Occludin. In addition, we identified that knockdown of Lnc00462717 or overexpression of miR-186-5p increased the accumulation of doxorubicin (Dox) in brain glioma via the ultrafast liquid chromatography-mass spectrometry system (UFLC-MS/MS system) and decreased the intracranial glioma volume in BALB/c nude mice. Taken together, these results show a novel molecular pathway in BTB that may provide a potential innovative strategy for glioma therapy.

Rapid quantitation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in rat urine using ultra-fast liquid chromatography mass spectrometry (UFLC/MS/MS)

ABSTRACTThe tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a component of tobacco smoke and is rapidly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Limited information is available on the relative systemic exposures resulting from NNK administration via the oral, intraperitoneal injection, and inhalation routes. Moreover, there is a need for a rapid method for simultaneous quantitative analyses of NNK and NNAL in rat urine. We developed a method based on Ultra Fast Liquid Chromatography Mass Spectrometry (UFLC/MS/MS) for the extraction and analysis of the potent lung carcinogens NNK and NNAL. Following addition of synthetic labeled internal standards, urine was introduced to 96 well plate Evolute® Express CX 30 mg solid phase extraction system. The eluates were dried under vacuum and reconstituted in mobile phase before injecting to the LC system. The use of UFLC allowed for a 7.1 min run time. The precision and accuracy of the samples was 1.2-6.6% relative standard deviation (%RSD) and 91-113% of the concentration added, respectively. The limits of detection for NNK and NNAL were 70 and 3.0 pg/mL, respectively. The selectivity and sensitivity of this method improves the ability to measure these compounds at low concentrations and greatly facilitate toxicological studies of the NNK and NNAL.

Determination of oestrogen hormones in raw and treated water samples by reverse phase ultra-fast liquid chromatography mass spectrometry – a case study in Johannesburg South, South Africa

Hormones in water samples have been classified as ‘emerging pollutants’ and may pose a potential risk for humans. Hormones can be found in both surface and ground water at low concentrations. These compounds enter water streams through wastewater treatment plants (WWTP) and may elicit endocrine disruption to organisms. As a result of the low concentrations, a suitable analytical methodology is essential for their determination. In the past, residues of oestrogens in aqueous and solid environmental samples have commonly been analysed by gas chromatography (GC) equipped with mass spectrometry (MS). However, the necessary derivatisation steps are not only time consuming and laborious but also tedious. The first part of this case study was to develop a method to determine trace concentrations of the estrone (E1), 17α-estradiol (E2α), 17-β-estradiol (E2β) and 17-α-ethinylestradiol (EE2) hormones using reverse phase ultra-fast liquid chromatography mass spectrometry (RP-UFLC). Using the developed method, the second part of the case study was to determine the concentrations of the hormones in raw and treated potable water samples from the Vaal River catchment area in the south of Johannesburg, South Africa. Analytes were extracted by solid phase extraction (SPE C18 sorbent) cartridges and subsequently injected into ultra-fast liquid chromatography with the reverse phase column coupled to mass spectrometry (UFLC-MS) for analyte determinations. The limits of quantification were in the range of 0.24 to 0.32 ng·L-1 for the analytes. Accuracy was 95.6, 93.8, 97.6 and 100.9% for EE2, E2α, E2β and E1, respectively. In raw water samples taken during the rainy wet season, estrone was detected at concentrations of 0.90 and 4.43 ng·L-1. However, treated potable water samples did not contain detectable levels of hormones. This is potentially due to the ability of a water treatment process to remove endocrine disruptors during water purification.

Evaluation of pharmacokinetics and blood-brain barrier permeability of mitragynine using in vivo microdialysis technique

A microdialysis system coupled with a sensitive ultra-fast liquid chromatography–mass spectrometry (UFLC-MS) method was developed for the pharmacokinetic analysis of mitragynine in rat blood and striatum. Mitragynine is an active alkaloid of Mitragyna speciosa and has been proposed to be used for opioid withdrawal therapy. In this study, chromatographic separation was performed in a gradient elution mode with 0.1% formic acid and acetonitrile on a Zorbax Eclipse C18 column. The mass spectrometric (MS) analysis was carried out in a positive electrospray mode and mitragynine ion (m/z 399.2) was monitored in extracted ion chromatography. A good linearity range was obtained from 10-1000ng/mL with acceptable accuracy and precision parameters. The microdialysate was collected simultaneously from the striatum and the right jugular vein using microdialysis probes. After a single intravenous administration of 10mg/kg mitragynine, mitragynine showed a two-compartmental drug elimination pattern with half-life (T1/2) of approximately 13h. The percent of AUCbrain/AUCplasma of mitragynine was calculated and shown to be 65.8±4.5%. The results indicated that mitragynine could be a suitable molecule to develop into an opioid replacement drug based on its ideal pharmacokinetic properties, namely, small molecular size, lipophilic in nature and with excellent blood–brain barrier (BBB) permeability.

Tenuifolin, a saponin derived from Radix Polygalae, exhibits sleep-enhancing effects in mice

BackgroundRadix Polygalae, the dried root of Polygala tenuifolia, has been extensively used as a traditional Chinese medicine for promoting intelligence and tranquilization. Polygalasaponins extracted from the root of P. tenuifolia possess evident anxiolytic and sedative-hypnotic activities. Previous studies have reported that tenuifolin was a major constituent of polygalasaponins. PurposeThe currently study aims to investigate the hypnotic effect and possible mechanism of tenuifolin in freely moving mice. Design/MethodsThe hypnotic effects of tenuifolin (20, 40 and 80mg/kg, p.o.) were assessed by electroencephalographic (EEG) and electromyographic (EMG) analysis. Double-staining immunohistochemistry test was performed to evaluate the neuronal activity of sleep-wake regulating brain areas. High performance liquid chromatograph- electrochemical detection (HPLC-ECD) and ultrafast liquid chromatography−mass spectrometry (UFLC-MS) were used for the detection of neurotransmitters. Locomotor activity was measured by Open-field Test. ResultsTenuifolin at doses of 40 and 80mg/kg (p.o.) significantly prolonged the total sleep time by increasing the amount of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep, associated with the significant increase in the bouts of episodes respectively. After administration of tenuifolin, the cortical EEG power spectral densities during NREM and REM sleep were similar to that of natural sleep (vehicle) and thus compatible with physiological sleep. Double-immunohistochemistry staining test showed that tenuifolin increased the c-Fos positive ratios of GABAergic NREM sleep-promoting neurons in ventrolateral preoptic area (VLPO), cholinergic REM sleep-promoting neurons in laterodorsal tegmental area (LDT) and pontomesencephalic tegmental area (PPT) and decreased the c-Fos positive ratios in wake-promoting neurons (locus coeruleus (LC) and perifornical area (Pef)). Neurotransmitter detections revealed that tenuifolin significantly reduced the noradrenaline (NA) levels in LC, VLPO, PPT and LDT, elevated the GABA levels in VLPO, LC and Pef and increased the acetylcholine (Ach) levels in LDT and PPT. In addition, tenuifolin did not cause any change to locomotor activity. ConclusionTaken together, these results provide the first experimental evidence of the significant sleep-enhancing effect of tenuifolin in mice. This effect appears to be mediated, at least in part, by the activation of GABAergic systems and/or by the inhibition of noradrenergic systems. Moreover, this study adds new scientific evidence and highlights the therapeutic potential of the medicinal plant P. tenuifolia in the development of phytomedicines with hypnotic properties.

Mass spectrometric evaluation of neurotransmitter levels in IMR 32 cell line in response to Ayurvedic medicines.

Ayurvedic herbal medicines are administered as part of disease management for many neurodevelopmental disorders like Autism Spectrum Disorder (ASD) and linked comorbid challenges. The biochemistry of the behavioral abnormalities as observed in comorbid conditions is already reported to involve neurotransmitters like gamma-aminobutyric acid (GABA), serotonin (5-HT) and dopamine (DA). The aim of our study is to evaluate the effect of ayurvedic medicines on neurotransmitter levels in IMR 32. Such a study will give some insight into the molecular mechanism of the action of these medicines and help us to understand their contributions in neurotransmitter homeostasis. Solutions of Brahmi, Brahmi vati, Brahmi ghrita and Saraswata ghrita, each at 50μM, were added to differentiated IMR 32 cells and grown for 24h. The cell secretion was analysed by ultra-fast liquid chromatography/mass spectrometry (UFLC/MS) in electrospray ionisation (ESI) mode for the neurotransmitters DA, 5-HT and GABA. The mobile phase selected was 0.1% formic acid with 15 μg/mL Na2 -EDTA (A) and 0.1% formic acid in acetonitrile (B) introduced in the ratio of 92:8. All neurotransmitters under study were eluted within 7min with GABA eluting at 3.82min, 5-HT at 4.48min and DA at 5.47min, respectively. Linearity was excellent with a correlation coefficient (R(2) ) of 0.999; repeatability and accuracy were also within acceptable range. All herbal drugs evaluated increased the neurotransmitter levels and Brahmi vati increased the neurotransmitter levels to a larger extent. Decreased levels of neurotransmitters were observed in behavioral abnormalities which were also observed in children with ASD. Herbal medicines given as part of ayurvedic medicine increased the neurotransmitter levels in IMR 32. Thus, these ayurvedic medicines when prescribed to children with ASD might alleviate the abnormal behavioral symptoms by maintaining neurotransmitter homeostasis. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of the protective effect of Zhi-Zi-da-Huang decoction on acute liver injury with cholestasis induced by α-naphthylisothiocyanate in rats

Ethnopharmacological relevanceZhi-Zi-Da-Huang decoction (ZZDHD), a classic traditional Chinese medicine (TCM) formula composed of four herbal medicines, has been widely used to treat various hepatobiliary disorders for a long time in China. However, the pharmacological effect of ZZDHD on liver injury with cholestasis is unrevealed. Aim of the studyTo investigate the hepatoprotective effect of ZZDHD against α-naphthylisothiocyanate (ANIT)-induced liver injury with cholestasis in rats. Materials and methodsThe rats were intragastrically (i.g.) given ZZDHD at doses of 1, 2 and 4g/kg (crude drug/body weight) once a day for seven days and treated with ANIT (75mg/kg via i.g.) to cause liver injury at 12h after the fifth administration. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltranspeptidase (γ-GTP), total bilirubin (TBIL), direct bilirubin (DBIL) and total bile acid (TBA), as well as bile flow were measured at 48h after ANIT treatment to evaluate the protective effect of ZZDHD. Moreover, the possible protective mechanisms were elucidated by assays of liver enzyme activities and component contents including malondialdehyde (MDA), myeloperoxidase (MPO), lipid peroxide (LPO), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). The biochemical observations were supplemented by histopathological examination. Ultra fast liquid chromatography–mass spectrometry (UFLC–MS) was used for the phytochemical analysis of ZZDHD. ResultsThe high dose (4g/kg) and middle dose (2g/kg) of ZZDHD exhibited significant and dose-dependent protective effect on ANIT-induced liver injury with cholestasis by reversing the changes in bile flow, the serum and hepatic enzymes, and histopathology of the liver tissue. Meanwhile, it was found that the low dose (1g/kg) of ZZDHD did not improve the biochemical indexes except serum TBIL, DBIL and TBA, which showed little protective effect. Phytochemical analysis revealed the presence of sixteen compounds in ZZDHD. ConclusionsThis study indicates that ZZDHD exerted a hepatoprotective effect on ANIT-induced liver injury with cholestasis in rats, and the mechanism of this activity is possibly related to its antioxidant properties.

Extraction and fractionation of phenolic acids and glycoalkaloids from potato peels using acidified water/ethanol-based solvents

Potato processing generates potato peels as byproducts. Methanolic extracts from the peels result in mixtures of phenolic acids and glycoalkaloids. Phenolic acids have potential for food applications owing to their antioxidant and antibacterial properties. However, when extracted from potatoes, their separation from toxic glycoalkaloids is needed prior to their applications in foods. Moreover, glycoalkaloids may be used as feedstock for synthesis of pharmaceuticals. This study aimed to develop a method for the extraction and fractionation of phenolic acids and glycoalkaloids from potato peels using food grade water/ethanol-based solvents. Samples were analyzed by ultrafast liquid chromatography (UFLC) and/or ultrafast liquid chromatography–mass spectrometry (UFLC–MS). A methanol-based solvent for extraction was used as a control to be compared with two aqueous ethanolic solvents acidified with acetic acid. The recovery of the predominant compounds from potato peels was comparable for all three solvents. Extraction yielded per 100g of potato peel fresh weight 17.0mg α-chaconine, 7.1mg α-solanine, 0.1mg solanidine, 4.8mg caffeic acid, 13.3mg neochlorogenic acid, and 77.6mg chlorogenic acid. More than 90% of these compounds were recovered after two consecutive extractions. The crude extract was fractionated by solid-phase extraction at pH7 and eluted with aqueous ethanol. Quantitative recovery of the phenolic acids and glycoalkaloids was achieved in their corresponding fractions. Hydrolysis followed by solid-phase fractionation of the crude extract allowed recovery of 139μmol caffeic acid/100g potato peel fresh weight. Partial degradation of caffeic acid and glycoalkaloids occurred during the process. Degradation of caffeic acid can be likely mitigated by the addition of antioxidants and metal chelators. The method developed in this study allows the sustainable recovery of secondary plant metabolites from potato peels and their fractionation using food grade water/ethanolic solvents for application of phenolic extracts free of toxic glycoalkaloids for food preservation, and of glycoalkaloid extracts for synthesis of pharmaceuticals.

Quantification of branched-chain keto acids in tissue by ultra fast liquid chromatography–mass spectrometry

Branched-chain keto acids (BCKAs) are associated with increased susceptibility to several degenerative diseases. However, BCKA concentrations in tissues or the amounts of tissue available are frequently at the limit of detection for standard plasma methods. To accurately and quickly determine tissue BCKAs, we have developed a sensitive ultra fast liquid chromatography–mass spectrometry (UFLC–MS) method. BCKAs from deproteinized tissue extractions were o-phenylenediamine (OPD) derivatized, ethyl acetate extracted, lyophilized in a vacuum centrifuge, and reconstituted in 200mM ammonium acetate. Samples were injected onto a Shimadzu UFLC system coupled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument that detected masses of the OPD BCKA products using a multiple reaction monitoring method. An OPD-derivatized 13C-labeled keto acid was used as an internal standard. Application of the method for C57BL/6J (wild-type) and PP2Cm knockout mouse tissues, including kidney, adipose tissue, liver, gastrocnemius, and hypothalamus, is shown. The lowest tissue concentration measured by this method was 20nM, with the standard curve covering a wide range (7.8–32,000nM). Liquid chromatography–mass spectrometry run times for this assay were less than 5min, facilitating high throughput, and the OPD derivatives were found to be stable over several days.

Determination of phthalate esters in physiological saline solution by monolithic silica spin column extraction method

Monolithic silica spin column extraction (MonoSpin-SPE) was developed as a simple, sensitive, and eco-friendly pretreatment method which combined with ultra-fast liquid chromatography-mass spectrometry (UFLC-MS) to determine the levels of six phthalate esters, dimethyl-(DMP), diethyl-(DEP), dipropyl- [DPrP], butyl-benzyl-(BBP), dicyclohexyl(DcHP), and di- n-octyl-(DOP) phthalate in physiological saline samples. Under optimized experimental conditions, the method was linear in the following ranges: 0.2- 50μ/L for DMP, DEP, DPrP, DcHP and DOP; 5 – 100μ/L for BBP. The correlation coefficients (R2) were in the range of O. 9951 – O. 9995 for all the analytes and the limits of detection (LODs) and limits of quantification (LOQs) were in the ranges of 0.02 – 0.9μ/L and 0.08 – 2.7μ/L, respectively. The pretreatment process showed good reproducibility with inter-day and intra-day relative standard deviations (RSDs) below 8.5% and 11.2%, respectively. This method was used to determine the levels of six phthalate esters in physiological saline samples and the recoveries ranged from 71.2% to 107. 3%. DMP and DEP were found in actual physical saline samples (brand A and brand B).

Lipidomics Analysis Reveals Efficient Storage of Hepatic Triacylglycerides Enriched in Unsaturated Fatty Acids after One Bout of Exercise in Mice

BackgroundEndurance exercise induces lipolysis, increases circulating concentrations of free fatty acids (FFA) and the uptake and oxidation of fatty acids in the working muscle. Less is known about the regulation of lipid metabolism in the liver during and post-exercise.Methodology/Principal FindingsWe performed an ultra fast liquid chromatography-mass spectrometry (UFLC-MS) based lipidomics analysis of liver tissue samples obtained from C57Bl/6J mice immediately after a 60 min treadmill run of moderate intensity, and after 3 h of recovery. The PLS-DA scores plot for 115 quantified lipid molecular species revealed a clear separation of the hepatic lipid profile of sedentary from recovering mice, but not from mice immediately after running. 21 lipid species were considered to be most responsible for the difference in the hepatic lipid profiles, including 17 triacylglycerides (TG), one lysophosphatidylcholine (LPC) and three phosphatidylcholines (PC). TG species were found to be more abundant in the recovery phase, while PC species were decreased. The degree of accumulation of individual TG species correlated well with the amount of theoretical energy stored whereas no increase was found for TG species containing only saturated or one monounsaturated fatty acid. Total liver TG content as assayed by an enzymatic method was increased to 163% in the recovery phase, while it was significantly decreased in skeletal muscle by the exercise bout and remained less in the recovery phase. Results from fasted and refed mice indicate that fasting-induced lipolysis was associated with a pronounced accumulation of hepatic TG, which is reversed by refeeding for 5 h. Thus food intake per se did not elevate hepatic TG.ConclusionThese data indicate that high availability of FFA induced by endurance exercise or fasting resulted in a transient hepatic TG accumulation, while muscle TG content was decreased during exercise presumably due to increased muscle fatty acid oxidation.

Identification and quantification of sophorolipid analogs using ultra-fast liquid chromatography–mass spectrometry

An ultra-fast liquid chromatographic method combined with atmospheric pressure chemical ionization mass detection (UHPLC/APCI-MS) has been developed for the separation and quantification of sophorolipid analogs produced by the yeast Candida bombicola. The sophorolipid mixture was produced by growing the yeast in the presence of glucose and oleic acid under higher aeration. It was found that more than 95% of the analogs are lactonic sophorolipids and all the produced sophorolipids produced were either mono- or di-acetylated. Also observed was a sophorolipid analog with a tri-unsaturated fatty acid, which has not been reported previously.