Sildenafil and its analogues (tadalafil and vardenafil) are phosphodiesterase type 5 inhibitors used in the treatment of male erectile dysfunction. Some dietary supplements, herbal preparations and food products which claim to enhance male sexual function have been found to be adulterated with these drugs. In this study, a gas chromatograph-mass spectrometer (GC-MS) assay was developed for identification of the drugs. In addition to good and short chromatographic separation that can be achieved within 6 min by using a short 10 m capillary column, no prior sample clean-up before GC-MS analysis was required, thus making this assay a cost saving and rapid method. Furthermore, the assay is specific as the identification of sildenafil, tadalafil and vardenafil were done by detection of molecular ions; m/z 474, 389 and 488, [corrected] respectively, and several other characteristic ions resulted from the mass fragmentation of individual molecules. Using our currently developed assay, sildenafil and its analogues were successfully identified in food and herbal matrices.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry/methods*
A novel microextraction technique termed solid phase membrane tip extraction (SPMTE) was developed. Selected triazine herbicides were employed as model compounds to evaluate the extraction performance and multiwall carbon nanotubes (MWCNTs) were used as the adsorbent enclosed in SPMTE device. The SPMTE procedure was performed in semi-automated dynamic mode and several important extraction parameters were comprehensively optimized. Under the optimum extraction conditions, the method showed good linearity in the range of 1-100 microg/L, acceptable reproducibility (RSD 6-8%, n=5), low limits of detection (0.2-0.5 microg/L), and satisfactory relative recoveries (95-101%). The SPMTE device could be regenerated and reused up to 15 analyses with no analyte carry-over effects observed. Comparison was made with commercially available solid phase extraction-molecular imprinted polymer cartridge (SPE-MIP) for triazine herbicides as the reference method. The new developed method showed comparable or even better results against reference method and is a simple, feasible, and cost effective microextraction technique.
An anion exchange monolithic silica capillary column was prepared by surface modification of a hybrid monolithic silica capillary column prepared from a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS). The surface modification was carried out by on-column copolymerization of N-[3-(dimethylamino)propyl]acrylamide methyl chloride-quaternary salt (DMAPAA-Q) with 3-methacryloxypropyl moieties bonded as an anchor to the silica surface to form a strong anion exchange stationary phase. The columns were examined for their performance in liquid chromatography (LC) and capillary electrochromatography (CEC) separations of common anions. The ions were separated using 50 mM phosphate buffer at pH 6.6. Evaluation by LC produced an average of 30,000 theoretical plates (33 cm column length) for the inorganic anions and nucleotides. Evaluation by CEC, using the same buffer, produced enhanced chromatographic performance of up to ca. 90,000 theoretical plates and a theoretical plate height of ca. 4 mum. Although reduced efficiency was observed for inorganic anions that were retained a long time, the results of this study highlight the potential utility of the DMAPAA-Q stationary phase for anion separations.
A simple and sensitive RP-HPLC-UV method was developed and validated for simultaneous determination of atenolol and propranolol and subsequently applied to investigate the effect of dimethyl sulfoxide in rat in situ intestinal permeability studies. Atenolol (400 microm) and propranolol (100 microm) were perfused in the small intestine of anaesthetized (pentobarbitone sodium 60 mg/kg, i.p.) male Sprague-Dawley rats either in the presence (1, 3 and 5%) or in the absence of dimethyl sulfoxide. There was no significant alteration (p > 0.05) in the permeability of atenolol and propranolol, which indicated there was no effect of various concentrations of dimethyl sulfoxide (1-5%) on the membrane integrity of the rat intestinal tissues. The analytical method was validated on a C(4) column with a mobile phase comprising ammonium acetate buffer (pH 3.5, 0.02 m) and acetonitrile in the ratio of 30:70 (v/v) at a flow rate of 1.0 mL/min. The validated method was found to be accurate and precise and stability studies were carried out at different storage conditions and both analytes were found to be stable. These findings are applicable for determining the absorbability of water-insoluble drugs and new chemical entities for the purpose of classifying them in the biopharmaceutical classification system.
Matched MeSH terms: Chromatography, High Pressure Liquid/methods*
A simple, sensitive and specific reversed-phase high-performance liquid chromatographic method with UV detection at 251 nm was developed for quantitation of buparvaquone (BPQ) in human and rabbit plasma. The method utilizes 250 microL of plasma and sample preparation involves protein precipitation followed by solid-phase extraction. The method was validated on a C18 column with mobile phase consisting of ammonium acetate buffer (0.02 m, pH 3.0) and acetonitrile in the ratio of 18:82 (v/v) at a flow rate of 1.1 mL/min. The calibration curves were linear (correlation coefficient>or=0.998) in the selected range. The method is specific and sensitive with limit of quantitation of 50 ng/mL for BPQ. The validated method was found to be accurate and precise in the working calibration range. Stability studies were carried out at different storage conditions and BPQ was found to be stable. Partial validation studies were carried out using rabbit plasma and intra- and inter-day precision and accuracy were within 7%. This method is simple, reliable and can be routinely used for preclinical pharmacokinetic studies for BPQ.
Matched MeSH terms: Chromatography, High Pressure Liquid/methods*
A new sample pre-treatment technique termed cone-shaped membrane liquid phase microextraction (CSM-LPME) was developed and combined with micro-liquid chromatography (micro-LC) for the determination of selected pesticides in water samples. Four pesticides (hexaconazole, procymidone, quinalphos and vinclozolin) were considered as target analytes. Several important extraction parameters such as types of extraction solvent, agitation rate, pH value, total exposure time and effect of salt and humic acids were optimized. Enrichment factors of > 50 folds were easily achieved within 20 min of extraction. The analytical data demonstrated relative standard deviations for the reproducibility of the optimized CSM-LPME method ranging from 6.3 to 7.5%. The correlation coefficients of the calibration curves were at least 0.9995 across a concentration range of 2-100 microg/L. The detection limits for all the analytes were found to be in the range of 1.1-1.9 microg/L.
In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
Poly(oligo(ethylene glycol) methacrylate) (POEGMA), an inert polymer was grafted onto an anion exchange adsorbent for the exclusion of relatively larger hepatitis B virus-like particles (HB-VLPs) from the anion exchange ligand (Q) and at the same time this process allowed the selective adsorption of smaller size Escherichia coli host cell proteins (HCPs). The chain lengths of the POEGMA grafted were modulated by varying the amount of monomers used in the polymer grafting. The purification factor and yield of the HB-VLPs obtained from the flow-through of negative chromatography were 2.3 and 66.0±3.1%, respectively, when shorter chain length of POEGMA (SQ) was grafted. Adsorbent grafted with longer chain of POEGMA (LQ) excluded some HCPs that are larger in size together with the HB-VLPs, reducing the purity of the recovered HB-VLPs. Further heat-treatment of the flow-through pool from SQ followed by centrifugation increased the purity of heat stable HB-VLPs to 87.5±1.1%. Heat-treatment of the flow through sample resulted in thermal denaturation and aggregation of HCPs, while the heat stable HB-VLPs still remained intact as observed under a transmission electron microscope. The performance of the negative chromatography together with heat treatment in the purification of HB-VLPs is far better than the reported bind-and-elute techniques.
Matched MeSH terms: Chromatography, Ion Exchange/methods
Temperature is one of the key factors in limiting the distribution of plants and controlling major metabolic processes. A series of simulated reciprocal transplant experiments were performed to investigate the effect of temperature on plant chemical composition. Polygonum minus of different lowland and highland origin were grown under a controlled environment with different temperature regimes to study the effects on secondary metabolites. We applied gas chromatography-mass spectrometry and liquid chromatography time-of-flight mass spectrometry to identify the chemical compounds. A total of 37 volatile organic compounds and 85 flavonoids were detected, with the largest response observed in the compositional changes of aldehydes and terpenes in highland plants under higher temperature treatment. Significantly less anthocyanidin compounds and larger amounts of flavonols were detected under higher temperature treatment. We also studied natural variation in the different plant populations growing under the same environment and identified compounds unique to each population through metabolite fingerprinting. This study shows that the origin of different plant populations influences the effects of temperature on chemical composition.
Matched MeSH terms: Chromatography, Liquid; Gas Chromatography-Mass Spectrometry
Colorectal cancer (CRC) is one of the most common human malignant tumors worldwide. Arising from the transformation of epithelial cells in the colon and/or rectum into malignant cells, the foundation of CRC pathogenesis lies in the progressive accumulation of mutations in oncogenes and tumor-suppressor genes, such as APC and KRAS. Resistance to apoptosis is one of the key mechanisms in the development of CRC as it is for any other kind of cancer. Natural products have been shown to induce the expression of apoptosis regulators that are blocked in cancer cells. In the present study, a series of in vitro assays were employed to study the apoptosis inducing attributes of Isoledene rich sub-fraction (IR-SF) collected from the oleo-gum resin of M. ferrea. Data obtained, shows that IR-SF inhibited cell proliferation and induced typical apoptotic changes in the overall morphology of all the CRC cell lines tested. Fluorescent staining assays revealed characteristic nuclear condensation, and marked decrease in mitochondrial outer membrane potential in treated cells. In addition, an increment in the levels of ROS, caspase-8,-9 and -3 was observed. Proteomic analysis revealed that IR-SF up-regulated the expression of pro-apoptotic proteins, i.e., Bid, Bid and cytochrome c. Cytochrome c in turn activated caspases cascade resulting in the induction of apoptosis. Moreover, IR-SF significantly down-regulated Bcl-2, Bcl-w, survivin, xIAP and HSPs pro-proteins and induced DNA fragmentation and G0/G1-phase arrest in HCT 116 cells. Chemical characterization of IR-SF by GC-MS and HPLC methods identified Isoledene as one of the major compounds. Altogether, the results of the present study demonstrate that IR-SF may induce apoptosis in human colorectal carcinoma cells through activation of ROS-mediated apoptotic pathways.
Matched MeSH terms: Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry
C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.
Matched MeSH terms: Chromatography, Affinity; Chromatography, Gel
A novel derivative of sucrose, beta-(3,6-di-O-feruloyl)-fructofuranosyl-alpha-(2,3,4,6-tetra-O-ac etyl)- glucopyranoside, was isolated from the wood of Bhesa paniculata. Its structure was determined by a combination of 2D 1H-1H and 1H-13C correlation NMR spectroscopy. The known compounds, glycerol 1-9',12'-octadecadienoate, beta-sitosterol, (+/-)-pinoresinol, methyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxybenzoic acid, anofinic acid and 2-(1'-methylethenyl)-benzofuran-5-carboxylic acid were also isolated.
Matched MeSH terms: Chromatography, High Pressure Liquid/methods
Peptides with biological properties, that is, bioactive peptides, are a class of biomolecules whose health-promoting properties are increasingly being exploited in food and health products. However, research on targeted techniques for the detection and quantification of these peptides is still in its infancy. Such information is needed in order to enhance the biological and chemometric characterization of peptides and their subsequent application in the functional food and pharmaceutical industries. In this review, the role of classic techniques such as electrophoretic, chromatographic, and peptide mass spectrometry in the structure-informed detection and quantitation of bioactive peptides are discussed. Prospects for the use of aptamers in the characterization of bioactive peptides are also discussed. PRACTICAL APPLICATIONS: Although bioactive peptides have huge potential applications in the functional foods and health area, there are limited techniques in enhancing throughput detection, quantification, and characterization of these peptides. This review discusses state-of-the-art techniques relevant in complementing bioactive detection and profiling irrespective of the small number of amino acid units. Insights into challenges, possible remedies and prevailing areas requiring thorough research in the extant literature for food chemists and biotechnologists are also presented.
Roasting is an important process in cocoa production which may lead to formation of non-desirable compounds such as polycyclic aromatic hydrocarbons (PAHs). Therefore, PAH4 (sum of four different polycyclic aromatic hydrocarbons; benz[a]anthracene, chrysene, benzo[b]fluoranthene, and benzo[a]pyrene) in roasted cocoa beans was determined using a modified method (combination of QuEChERS and DLLME), and quantified by HPLC-FLD. The modified method was validated and met the performance criteria required by the EU Regulation (No. 836/2011). Results show a significant (p
Matched MeSH terms: Chromatography, High Pressure Liquid/methods*
Mitragyna speciosa (Kratom) is currently used as a drug of abuse. When monitoring its abuse in urine, several alkaloids and their metabolites must be considered. In former studies, mitragynine (MG), its diastereomer speciogynine (SG), and paynantheine and their metabolites could be identified in rat and human urine using LC-MS(n). In Kratom users' urines, besides MG and SG, further isomeric compounds were detected. To elucidate whether the MG and SG diastereomer speciociliatine (SC) and its metabolites represent further compounds, the phase I and II metabolites of SC were identified first in rat urine after the administration of the pure alkaloid. Then, the identified rat metabolites were screened for in the urine of Kratom users using the above-mentioned LC-MS(n) procedure. Considering the mass spectra and retention times, it could be confirmed that SC and its metabolites are so far the unidentified isomers in human urine. In conclusion, SC and its metabolites can be used as further markers for Kratom use, especially by consumption of raw material or products that contain a high amount of fruits of the Malaysian plant M. speciosa.
Background: Cervical cancer is one of the most commonly diagnosed neoplasms and a leading cause of cancer death among females worldwide. Limitations with conventional medical treatments have driven researchers to search for alternative approaches using natural products. This study aimed to detemine potential anti-proliferative effects of methanol and water extracts of Pyrrosia piloselloides (P. piloselloides) on the HeLa cell line. Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to determine IC50 concentrations and apoptosis analysis was by flow cytometry. To identify chemical compounds in the extracts, gas chromatography-mass spectrometry (GC-MS) was employed. Results: P. piloselloides methanol extracts (PPME) showed antiproliferative effects on HeL awith an IC50 of 16.25μg/mL while the P. piloselloides water extract (PPWE) was without influence. Neither extract showed any significant effects on apoptosis. GC-MS analysis, revealed 5-hydroxymethylfurfural (23.1%), allopurinol (8.66%) and 3, 5-dihydroxy-6-methyl-2,3-dihydropyran-4-one (7.41%) as major components in the PPME, while sulfolan-3-ol (10.1%), linoleic acid (9.06%) and β-sitosterol acetate (7.98%) predominated in the PPWE case. Conclusion: This first study of P. piloselloides showed PPME to exert potent anti-proliferative effect on HeLa cell lines. Further research now needs to be performed to establish the mechanisms of inhibition.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry/methods
This study aims to elucidate the antiproliferative mechanism of hydroxychavicol (HC). Its effects on cell cycle, apoptosis, and the expression of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK) in HT-29 colon cancer cells were investigated. HC was isolated from Piper betle leaf (PBL) and verified by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and gas chromatography-mass spectrometry (GC-MS). The cytotoxic effects of the standard drug 5-fluorouracil (5-FU), PBL water extract, and HC on HT-29 cells were measured after 24, 48, and 72 h of treatment. Cell cycle and apoptosis modulation by 5-FU and HC treatments were investigated up to 30 h. Changes in phosphorylated JNK (pJNK) and P38 (pP38) MAPK expression were observed up to 18 h. The half maximal inhibitory concentration (IC50) values of HC (30 μg/mL) and PBL water extract (380 μg/mL) were achieved at 24 h, whereas the IC50 of 5-FU (50 μmol/L) was obtained at 72 h. Cell cycle arrest at the G0/G1 phase in HC-treated cells was observed from 12 h onwards. Higher apoptotic cell death in HC-treated cells compared to 5-FU-treated cells (P<0.05) was observed. High expression of pJNK and pP38 MAPK was observed at 12 h in HC-treated cells, but not in 5-FU-treated HT-29 cells (P<0.05). It is concluded that HC induces cell cycle arrest and apoptosis of HT-29 cells, with these actions possibly mediated by JNK and P38 MAPK.
Matched MeSH terms: Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry
The oil of Adenanthera pavonina L. seeds was analysed by chromatographic and instrumental means. The oil was found to be rich in neutral lipids (86.2%), and low in polar lipids (13.8%). The neutral lipids consisted mainly of triacylglycerols (64.2%). Unsaturated fatty acids were found as high as 71%, while the percentage of saturated fatty acids was only 29%. GC and GC/MS analyses revealed linoleic, oleic and lignocerotic acid to be predominant among all fatty acids in the A. pavonina oil, whereas stigmasterol was the major steroid identified within this study. Subsequently, the oil was used for preparation of submicron oil-in-water (o/w) lipid emulsions. Lipid emulsions were formulated by using soybean lecithin (SL) to investigate their particle size, Zeta potential and stability at the different oil and SL ratios. The results obtained indicate possible applications of the tested oil in pharmaceutical and medical fields as drug and cosmetic active ingredient carriers.
2,4,6-trihydroxy-3-geranylacetophenone (tHGA) is a bioactive compound that shows excellent anti-inflammatory properties. However, its pharmacokinetics and metabolism have yet to be evaluated. In this study, a sensitive LC-HRMS method was developed and validated to quantify tHGA in rat plasma. The method showed good linearity (0.5-80 ng/mL). The accuracy and precision were within 10%. Pharmacokinetic investigations were performed on three groups of six rats. The first two groups were given oral administrations of unformulated and liposome-encapsulated tHGA, respectively, while the third group received intraperitoneal administration of liposome-encapsulated tHGA. The maximum concentration (Cmax), the time required to reach Cmax (tmax), elimination half-life (t1/2) and area under curve (AUC0-24) values for intraperitoneal administration were 54.6 ng/mL, 1.5 h, 6.7 h, and 193.9 ng/mL·h, respectively. For the oral administration of unformulated and formulated tHGA, Cmax values were 5.4 and 14.5 ng/mL, tmax values were 0.25 h for both, t1/2 values were 6.9 and 6.6 h, and AUC0-24 values were 17.6 and 40.7 ng/mL·h, respectively. The liposomal formulation improved the relative oral bioavailability of tHGA from 9.1% to 21.0% which was a 2.3-fold increment. Further, a total of 12 metabolites were detected and structurally characterized. The metabolites were mainly products of oxidation and glucuronide conjugation.
In this study, the microcrystalline cellulose/metal-organic framework 199 hybrid (MCC/MOF-199) was applied as sorbent for the dispersive micro-solid phase-extraction (D-μSPE) of chlorophenols. The D-μSPE method combined with high-performance liquid chromatography- ultraviolet detection (HPLC-UV) was employed to determine of four chlorophenols including 2-chlorophenol (2-CP), 4-chlorophenol (4-CP), 2,3-dichlorophenol (2,3-DCP), and 2,5-dichlorophenol (2,5-DCP) in aqueous. The main parameters of the D-μSPE process that influence the extraction (i.e. the amount of sorbent, elution condition, extraction time, and pH) were investigated and optimized. Based on the outputs, the presence of MCC on the surface of MOF-199 leads to improve the properties of MOF-199 and the MCC/MOF-199 has the highest sorption capacity, durability, and porosity in comparison with MCC and MOF-199. According to the validation study at the optimized conditions, the linearity for the analytes was achieved in the range from 0.1 to 200 ng mL-1 for 2-CP and 4-CP and 0.15 to 200 ng mL-1 for 2,3-DCP and 2,5-DCP with correlation coefficients between 0.9928 and 0.9965. The limits of detection calculated at S/N=3 were in the range of 0.03-0.05 ng mL-1. Besides, the relative standard deviations (RSDs) for three spiking levels (0.2, 10,100 ng mL-1) do not exceed 6.8% and extraction recoveries are between 81.0% and 88.3%. Finally, the D-μSPE-HPLC-UV method was successfully applied to the analysis of CPs in real water samples (mineral, river and wastewater samples) with good recoveries (95.8 to 99.5%) and satisfactory precisions (RSD < 6.8%).
Matched MeSH terms: Chromatography, High Pressure Liquid/methods