Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.
Matched MeSH terms: Chromatography, Affinity/methods; Chromatography, Ion Exchange/methods
The fatty acid composition and trans fatty acid (TFA) contents of biscuits products were determined by gas chromatography, using a highly polar 100m capillary column (HP-88) and flame ionization detection. Total TFA ranged from 0.00 – 0.52 g/100 g total fatty acids and 0.12 – 0.68 g/100 g total fatty acids for local packed and unpacked biscuits, respectively. In imported biscuits, total TFA was higher ranging from 0.03 – 3.09 g/100 g of total fatty acids. Trans 16:1 was the most abundant, with values ranging from 0.01% to 38% followed by trans 18:1 Δ11 (0.01% - 13.11%), trans 18:1 Δ9 (0.01% - 4.68%), trans 18:2 (0.23% - 2.77%) and small quantities of trans 18:1 Δ6. CLA, the natural TFA constituted from 0.1% to
In recent years, environmental concerns over ultra-trace levels of steroid estrogens concentrations in water samples have increased because of their adverse effects on human and animal life. Special attention to the analytical techniques used to quantify steroid estrogens in water samples is therefore increasingly important. The objective of this review was to present an overview of both instrumental and non-instrumental analytical techniques available for the determination of steroid estrogens in water samples, evidencing their respective potential advantages and limitations using the Need, Approach, Benefit, and Competition (NABC) approach. The analytical techniques highlighted in this review were instrumental and non-instrumental analytical techniques namely gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS), enzyme-linked immuno sorbent assay (ELISA), radio immuno assay (RIA), yeast estrogen screen (YES) assay, and human breast cancer cell line proliferation (E-screen) assay. The complexity of water samples and their low estrogenic concentrations necessitates the use of highly sensitive instrumental analytical techniques (GC-MS and LC-MS) and non-instrumental analytical techniques (ELISA, RIA, YES assay and E-screen assay) to quantify steroid estrogens. Both instrumental and non-instrumental analytical techniques have their own advantages and limitations. However, the non-instrumental ELISA analytical techniques, thanks to its lower detection limit and simplicity, its rapidity and cost-effectiveness, currently appears to be the most reliable for determining steroid estrogens in water samples.
Matched MeSH terms: Chromatography, Liquid/methods*; Gas Chromatography-Mass Spectrometry/methods*
Methyl esters from the triglyceride fraction of the neutral lipids of natural rubber latex were found by gas liquid chromatography to contain about 90% of a furanoid acid. Spectroscopic analysis identified the acid as 10,13-epoxy-11-methyloctadeca-10,12-dienoic acid.
The growth in driving force and popularity of cyclodextrin (CDs) and ionic liquids (ILs) as promising materials in the field of analytical chemistry has resulted in an exponentially increase of their exploitation and production in analytical chemistry field. CDs belong to the family of cyclic oligosaccharides composing of α-(1,4) linked glucopyranose subunits and possess a cage-like supramolecular structure. This structure enables chemical reactions to proceed between interacting ions, radical or molecules in the absence of covalent bonds. Conversely, ILs are an ionic fluids comprising of only cation and anion often with immeasurable vapor pressure making them as green or designer solvent. The cooperative effect between CD and IL due to their fascinating properties, have nowadays contributed their footprints for a better development in analytical chemistry nowadays. This comprehensive review serves to give an overview on some of the recent studies and provides an analytical trend for the application of CDs with the combination of ILs that possess beneficial and remarkable effects in analytical chemistry including their use in various sample preparation techniques such as solid phase extraction, magnetic solid phase extraction, cloud point extraction, microextraction, and separation techniques which includes gas chromatography, high-performance liquid chromatography, capillary electrophoresis as well as applications of electrochemical sensors as electrode modifiers with references to recent applications. This review will highlight the nature of interactions and synergic effects between CDs, ILs, and analytes. It is hoped that this review will stimulate further research in analytical chemistry.
Matched MeSH terms: Chromatography, Gas/methods; Chromatography, High Pressure Liquid/methods
Citrus peel essential oils have an impressive range of food and medicinal uses. In the present study we investigated the variation in the yield and chemical composition of the essential oils isolated from fresh, ambient-, and oven-dried peels of three Citrus species namely Citrus reticulata (C. reticulata), Citrus sinensis (C. sinensis) and Citrus paradisii (C. paradisii). The hydro-distilled essential oil content from fresh-, ambient-, and oven-dried peels of C. reticulata, C. sinensis and C. paradisii ranged from 0.30-0.50, 0.24-1.07 and 0.20-0.40 g/100 g, respectively. The maximum amount of the oil was determined in oven-dried while the minimum in fresh peel samples. Using GC and GC/MS, a total of 16-27, 17-24 and 18-40 chemical constituents were identified in the peel essential oils of C. reticulate, C. sinensis and C. paradisii, respectively. The content of limonene, the most prevalent chemical constituent, detected in these essential oils, ranged from 64.1-71.1% (C. reticulata), 66.8-80.9% (C. sinensis) and 50.8-65.5% (C. paradisii). The yield and content of most of the chemical components including limonene (the principal chemical compound detected) of the tested essential oils varied significantly (p < 0.05) with respect to drying treatments and species employed.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) has been recognised as an effective technique to analyse car paint. This study was conducted to assess the combination of Py-GC-MS and chemometric techniques to classify car paint primer, the inner layer of car paint system. Fifty car paint primer samples from various manufacturers were analysed using Py-GC-MS, and data set of identified pyrolysis products was subjected to principal component analysis (PCA) and discriminant analysis (DA). The PCA rendered 16 principal components with 86.33% of the total variance. The DA was useful to classify the car paint primer samples according to their types (1k and 2k primer) with 100% correct classification in the test set for all three modes (standard, stepwise forward and stepwise backward). Three compounds, indolizine, 1,3-benzenedicarbonitrile and p-terphenyl, were the most significant compounds in discriminating the car paint primer samples.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Entamoeba histolytica membrane proteins are important players toward the pathogenesis of amoebiasis, but the roles of most of the proteins are not fully understood. Since efficient protein extraction method is crucial for a successful MS analysis, three extractions methods are evaluated for the use in studying the membrane proteome of E. histolytica: Two commercial kits (ProteoExtract from Calbiochem and ProteoPrep from Sigma), and a conventional laboratory method. The results show that ProteoExtract and the conventional method gave higher protein yields compared to ProteoPrep. LC-ESI-MS/MS identifies 456, 482, and 551 membrane fraction proteins extracted using ProteoExtract, ProteoPrep, and a conventional method, respectively. In silico analysis predicts 108 (21%), 235 (45%), and 177 (34%) membrane proteins from the extracts of ProteoExtract, ProteoPrep, and the conventional method, respectively. Furthermore, analysis of the cytosolic and membrane fractions shows the highest selectivity of the membrane proteins using the ProteoPrep extraction kit. Overall, this study reports 828 E. histolytica membrane fraction proteins that include 249 predicted membrane proteins. The data are available via ProteomeXchange with identifier PXD010171.
This study explores the role of sterols as lipid biomarkers to indicate their input which originates from various sources in the marine environment. Sterols and their ratios were investigated in sediments taken from sixteen sampling stations at Pulau Tinggi, Johor in order to assess the sources of organic matter. The compounds extracted from the sediments were quantified using a gas chromatography-mass spectrometry (GC-MS). The distributions of sterols indicated that organic matter at all sampling stations originated from a mixture of marine source and terrestrial origins at different proportions. A total of eleven sterols were quantified, with the major compounds being phytosterols (44% of total sterols), cholesterol (11%), brassicasterol (11%) and fecal sterols (12%).
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Spillage of water polluting substances via industrial disaster may cause pollution to our environment. Thus, reversed-flow gas chromatography (RF-GC) technique, which applies flow perturbation gas chromatography, was used to investigate the evaporation and estimate the diffusion coefficients of liquid pollutants. Selected alcohols (99.9% purity) and its mixtures were used as samples. The evaporating liquids (stationary phase) were carried out by carrier gas-nitrogen, 99.9% purity (mobile phase) to the detector. The findings of this work showed the physicochemical measurements may vary depending on the composition of water and alcohol mixtures, temperature of the mixtures, as well as the types of alcohol used. This study implies that there is a variation in the results based on the concentration, types and temperature of the liquids that may contribute in the references for future research in the area of environmental pollution analysis.
Catalytic co-pyrolysis (CCP) of spent coffee ground (SCG) and cellulose over HZSM-5 and HY was characterized thermogravimetrically, and a catalytic pyrolysis of two samples was conducted using a tandem micro reactor that directly connected with gas chromatography-mass spectrometry. To access the more fundamental investigations on CCP, the effects of the zeolite pore structure, reaction temperature, in-situ/ex-situ reaction mode, catalyst to feedstock ratio, and the SCG and cellulose mixing ratio were experimentally evaluated. The temperature showing the highest thermal degradation rate of cellulose with SCG slightly delayed due to the interactions during the thermolysis of two samples. HZSM-5 in reference to HY produced more aromatic hydrocarbons from CCP. With respect to the reaction temperature, the formation of aromatic hydrocarbons increased with the pyrolytic temperature. Moreover, the in-situ/ex-situ reaction mode, catalyst/feedstock, and cellulose/SCG ratio were optimized to improve the aromatic hydrocarbon yield.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Capilliposide B (CPS-B) and Capilliposide C (CPS-C), as the key components in Lysimachia capillipes Hemsl., increasingly aroused the interest and research concern of many researchers due to the good bioactivities. Nowadays, the reference standards of CPS-B and CPS-C yield were very limited. Due to the deficit of reference standards, the determination could be difficult to carry out, and the quality control and evaluation would be restrained afterwards. To solve this urgent problem, a quantitative analysis of multi-components by single-marker (QAMS) method was proposed and established based on high-performance liquid-chromatography tandem evaporative light-scattering detector. In this QAMS method, the content of the two bioactive components could be calculated by buddlejasaponin IV, which is applied as an external standard and readily obtained. And the methodological experiments were evaluated and indicated accuracy, stability and feasibility of this QAMS method. Therefore, in this study, this built method would properly meet the requirement of determination of CPS-B, CPS-C and quality control of the L. capillipes Hemsl. plant.
Matched MeSH terms: Chromatography, High Pressure Liquid
Tocotrienol-rich fraction (TRF), a palm oil-derived vitamin E fraction, is reported to possess potent neuroprotective effects. However, the modulation of proteomes in differentiated human neuroblastoma SH-SY5Y cells (diff-neural cells) by TRF has not yet been reported. This study aims to investigate the proteomic changes implicated by TRF in human neural cells using a label-free liquid-chromatography-double mass spectrometry (LC-MS/MS) approach. Levodopa, a drug used in the treatment of Parkinson's disease (PD), was used as a drug control. The human SH-SY5Y neuroblastoma cells were differentiated for six days and treated with TRF or levodopa for 24 h prior to quantitative proteomic analysis. A total of 81 and 57 proteins were differentially expressed in diff-neural cells following treatment with TRF or levodopa, respectively. Among these proteins, 32 similar proteins were detected in both TRF and levodopa-treated neural cells, with 30 of these proteins showing similar expression pattern. The pathway enrichment analysis revealed that most of the proteins regulated by TRF and levodopa are key players in the ubiquitin-proteasome, calcium signalling, protein processing in the endoplasmic reticulum, mitochondrial pathway and axonal transport system. In conclusion, TRF is an essential functional food that affects differential protein expression in human neuronal cells at the cellular and molecular levels.
A method using solid phase extraction and liquid chromatography-tandem mass spectrometry to quantitatively detect mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine samples was developed and validated. The relevant metabolites were identified using multiple reaction monitoring in positive ionization mode using nalorphine as an internal standard. The method was validated for accuracy, precision, recovery, linearity, and lower limit of quantitation. The intra- and inter-day accuracy and precision were found in the range of 83.6-117.5% with coefficient of variation less than 13%. The percentage of recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine was within the range of 80.1-118.9%. The lower limit of quantification was 1 ng/mL for mitragynine, 2 ng/mL for 16-carboxy mitragynine, and 50 ng/mL for 9-O-demethyl mitragynine. The developed method was reproducible, high precision and accuracy with good linearity and recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine.
A simple analytical method for the determination of propionic acid and propionates in bakery products using a simple sample preparation procedure is described. The method involves the conversion of propionates to the non-ionized molecular form by adding glacial acetic acid, which is at the same time efficiently extracted into dichloromethane. After vortexing for 1 min, the extract was directly injected into a capillary gas chromatographic column with flame ionization detector. The method was applied for the determination of propionates in 112 commercial bakery samples. The levels of propionic acid plus propionates in bread, cake/ rolls, burger/hot dog buns and pita breads ranged from 197-1273, 98-1846, 546-1932 and 479-1680 µg mL -1 , respectively. No propionate was detected in any of the 36 biscuit samples analyzed.
In toxicological analysis, the analytical validation method is important to assess the exact risk of contaminants of emerging concern in the environment. Syringe filters are mainly used to remove impurities from sample solutions. However, the loss of analyte to the syringe filter could be considerable, causing an underestimate of the analyte concentrations. The current study develops and validates simultaneous liquid chromatography-mass spectrometry analysis using a direct filtration method to detect four groups of contaminants of emerging concern. The adsorption of the analyte onto three different matrices and six types of syringe filters is reported. The lowest adsorption of analytes was observed in methanol (16.72%), followed by deionized water (48.19%) and filtered surface lake water (48.94%). Irrespective of the type of the matrices, the lowest average adsorption by the syringe filter was observed in the 0.45 μm polypropylene membrane (15.15%), followed by the 0.20 μm polypropylene membrane (16.10%), the 0.20 μm regenerated cellulose (16.15%), the 0.20 μm polytetrafluoroethylene membrane (47.38%), the 0.45 μm nylon membrane (64.87%) and the 0.20 μm nylon membrane (71.30%). In conclusion, the recommended syringe filter membranes for contaminants of emerging concern analysis are polypropylene membranes and regenerated cellulose, regardless of the matrix used.
Adulteration of high-quality meat products using lower-priced meats, such as pork, is a crucial issue that could harm consumers. The consumption of pork is strictly forbidden in certain religions, such as Islam and Judaism. Therefore, the objective of this research was to develop untargeted metabolomics using liquid chromatography-high resolution mass spectrometry (LC-HRMS) combined with chemometrics for analysis of pork in beef meatballs for halal authentication. We investigated the use of non-targeted LC-HRMS as a method to detect such food adulteration. As a proof of concept using six technical replicates of pooled samples from beef and pork meat, we could show that metabolomics using LC-HRMS could be used for high-throughput screening of metabolites in meatballs made from beef and pork. Chemometrics of principal component analysis (PCA) was successfully used to differentiate beef meatballs and pork meatball samples. Partial least square-discriminant analysis (PLS-DA) clearly discriminated between halal and non-halal beef meatball samples with 100% accuracy. Orthogonal projection to latent structures-discriminant analysis (OPLS-DA) perfectly discriminated and classified meatballs made from beef, pork, and a mixture of beef-pork with a good level of fitness (R2X = 0.88, R2Y = 0.71) and good predictivity (Q2 = 0.55). Partial least square (PLS) and orthogonal PLS (OPLS) were successfully applied to predict the concentration of pork present in beef meatballs with high accuracy (R2 = 0.99) and high precision. Thirty-five potential metabolite markers were identified through VIP (variable important for projections) analysis. Metabolites of 1-(1Z-hexadecenyl)-sn-glycero-3-phosphocholine, acetyl-l-carnitine, dl-carnitine, anserine, hypoxanthine, linoleic acid, and prolylleucine had important roles for predicting pork in beef meatballs through S-line plot analysis. It can be concluded that a combination of untargeted metabolomics using LC-HRMS and chemometrics is promising to be developed as a standard analytical method for halal authentication of highly processed meat products.
A polymer inclusion membrane (PIM) based sampling probe was developed for electrokinetic extraction of drugs from biological fluids. The probe was fabricated by dip-coating a nonconductive glass capillary tube in a homogeneous PIM solution for three cycles. The PIM solution comprised cellulose triacetate (CTA), 2-nitrophenyl octyl ether (NPOE), and 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [EMIM][NTf2] in a ratio of 5:4:2. The developed probe electrokinetically extracted doxorubicin from human plasma, human serum, and dried blood spot (DBS). The practicability and reliability of the electrokinetic extraction were evaluated by LC-MS/MS to quantify the desorption of extracted doxorubicin. Under the optimized conditions, a quantification limit of 0.2-2 ng/mL was achieved for the three biological samples. The probe was further integrated into a portable battery-powered device for safe low-voltage (36 V) electrokinetic extraction. The developed technique is envisioned to provide a more efficient analytical workflow in the laboratory.
Shrimp meat is prone to autolysis and decay due to the abundance of endogenous enzymes and contamination from microorganisms. HVEF freezing can slow the spoilage of shrimp, producing small and uniform ice crystals, resulting in less damage to muscle tissue. In this study, HVEF technique was used to freeze the shrimp (Solenocera melantho), and the UPLC-MS metabolic technique was used to investigate the metabolites of frozen shrimp meat. Compared with the control group, 367 differential metabolites were identified in the HVEF group. Mapping them to the KEGG database, there were 108 with KEGG ID. Purine metabolism and pyrimidine metabolism were the most enriched pathways. In addition, phosphatidylcholines (PCs), inosine (HxR), and l-valine were identified as potential biomarkers associated with lipid, nucleotide, and organic acid metabolism, respectively. Overall, HVEF can improve freezing quality of shrimp meat by slowing down the metabolism of substances in the muscle of S. melantho.
In this study, the photocatalytic degradation of toxic pollutant (2-chlorophenol) in the presence of ZnO nanoparticles (ZnO NPs) was investigated under solar radiation. The three main factors, namely pH of solution, solar intensity and calcination temperature, were selected in order to examine their effects on the efficiency of the degradation process. The response surface methodology (RSM) technique based on D-optimal design was applied to optimise the process. ANOVA analysis showed that solar intensity and calcination temperature were the two significant factors for degradation efficiency. The optimum conditions in the model were solar intensity at 19.8 W/m(2), calcination temperature at 404 °C and pH of 6.0. The maximum degradation efficiency was predicted to be 90.5% which was in good agreement with the actual experimental value of 93.5%. The fit of the D-optimal design correlated very well with the experimental results with higher values of R (2) and R (2)adj correlation coefficients of 0.9847 and 0.9676, respectively. The intermediate mechanism behaviour of the 2-chlorophenol degradation process was determined by gas chromatography-mass spectrometry (GC-MS). The results confirmed that 2-chlorophenol was converted to acetic acid, a non-toxic compound.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry