Enzyme immobilization strategies for the design of robust and efficient biocatalysts

Different strategies for the preparation of efficient and robust immobilized biocatalysts are here reviewed. Different physico-chemical approaches are discussed.i.- The stabilization of enzyme by any kind of immobilization on pre-existing porous supports.ii.- The stabilization of enzymes by multipoint covalent attachment on support surfaces.iii.- Additional stabilization of immobilized-stabilized enzyme by physical or chemical modification with polymers.These three strategies can be easily developed when enzymes are immobilized in pre-existing porous supports. In addition to that, these immobilized-stabilized derivatives are optimal to develop enzyme reaction engineering and reactor engineering. Stabilizations ranging between 1000 and 100,000 folds regarding diluted soluble enzymes are here reported.     

UPLC-Q/TOF-MS coupled with multivariate analysis for comparative analysis of metabolomic in Dendrobium nobile from different growth altitudes

Dendrobium nobile alkaloids (DNLA) and glycosides are the main active components extracted from Dendrobium nobile Lindl. (D. nobile) used for thousands of years in China. The pharmacological effects of the above chemical components are significantly different. D. nobile is mainly grown at an altitude ranging from 230 to 800 m in Chishui City, Northwest Guizhou Province. However, it is unclear whether the metabolite in D. nobile is influenced by the planting altitude. Hence, to reveal the different metabolite in D. nobile cultivated at the altitude of 336 m, 528 m, and 692 m, ultra-high performance liquid chromatography with Q/TOF-MS couple with multivariate analysis were developed. Using the orthogonal partial least squares-discriminant analysis, 19 different metabolites were discovered and then tentatively assigned their structures as alkaloids and glycosides by comparing mass spectrometry data with in-house database and literature. Moreover, the result of semiquantitative analysis showed the content of dendrobine that was belonged to alkaloids significantly increased at the altitude of 692 m, whereas the content of glycosides demonstrated an accumulation trend at the altitude of 528 m. The results could provide valuable information for the optimal clinical drug therapeutics and provide a reference for quality control.     

Automated headspace solid‐phase microextraction and on‐fiber derivatization for the determination of clenbuterol in meat products by gas chromatography coupled to mass spectrometry

A method was developed for the determination of clenbuterol in meat using stable‐isotope‐dilution gas chromatography with mass spectrometry coupled with solid‐phase microextraction and on‐fiber derivatization. The samples were first homogenized with hydrochloric acid followed by protein deposition. After headspace solid‐phase microextraction and on‐fiber derivatization, the content of clenbuterol was measured with the aid of stable‐isotope dilution. The condition of solid‐phase microextraction was optimized by central composite design. The relative standard deviations, limit of detection, and recoveries for clenbuterol were 4.2–9.2%, 0.48 μg/kg, and 96–104%, respectively. The proposed method was satisfactory for analysis of real samples as compared with the Chinese standard method.     

Analysis of a new drug of abuse: Cathinone derivative 1‐(3,4‐dimethoxyphenyl)‐2‐(ethylamino)pentan‐1‐one

Recently, novel psychoactive drugs for human abuse such as amphetamines, phenethylamines, benzofuries, and tryptamines, cathinones have gained high popularity. These designer drugs are mainly sold via online stores as “bath salts” and are labeled “not for human consumption.” Due to the novelty of the compounds, only a little information about pharmacology, toxicology, and the long‐term damage they may cause is available. Moreover, there are only few analytical methods for their identification and analysis. Among new cathinone derivatives, 1‐(3,4‐dimethoxyphenyl)‐2‐(ethylamino)pentan‐1‐one (DL‐4662), became available via an internet shop. A sample of this compound was purchased and investigated. The first aim of our study was an identity check by NMR spectroscopy and gas chromatography with mass spectrometry. As many of the recreational drugs are chiral and are mainly sold as racemates, a further goal of our research was enantioseparation by gas chromatography with mass spectrometry and high‐performance liquid chromatography with UV detection, to prove whether DL‐4662 was traded enantiomerically pure or as racemic mixture. Both chiral separation methods showed the presence of a racemate.     

Determination of three kinds of banned drugs in milk powder by hybrid solid‐phase extraction purification combined with gas chromatography and mass spectrometry

A gradient clean‐up method for the quantification of five kinds of banned drugs (two hormones, two sedatives, and one chloramphenicol) in milk powder was developed. We used the combination of solid‐phase extraction purification with gas chromatography and mass spectrometry. Milk powder was initially hydrolyzed by β‐glucuronidase/arylsulfatase, and then the hydrolyzed solution was concentrated and purified using a C₈ and cation resin solid‐phase extraction column. To isolate hormones and chloramphenicol drugs, products from the previous step were diluted with methanol and further purified using a silica and diatomite solid‐phase extraction column. After derivatization, the drugs were analyzed by gas chromatography with mass spectrometry, and the hydrolyzed solution was diluted with 5% ammoniated methanol to purify sedatives before gas chromatography with mass spectrometry analysis. Results showed that after adding the banned drugs at concentrations of 0.3–10.0 μg/kg, the average recovery range was 78.2–97.3% with relative standard deviations of 5.3–12.5%. The limit of quantification of the banned drugs (S/N ≥ 10) was 0.3–5.0 μg/kg, whereas the limit of detection (S/N ≥ 3) was 0.1–2.0 μg/kg. The solid‐phase extraction gradient purification system was simple, rapid, and accurate, and could satisfy the detection requirements of hormone, sedatives, and chloramphenicol drugs when used together with gas chromatography and mass spectrometry.     

Screening anti‐tumor compounds from Ligusticum wallichii using cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry

Tyrosine 367 Cysteine‐fibroblast growth factor receptor 4 cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry was developed. Tyrosine 367 Cysteine‐HEK293 cells were used as the cell membrane stationary phase. The specificity and reproducibility of the cell membrane chromatography was evaluated using 1‐tert‐butyl‐3‐{2‐[4‐(diethylamino)butylamino]‐6‐(3,5‐dimethoxyphenyl)pyrido[2,3‐d]pyrimidin‐7‐yl}urea, nimodipine and dexamethasone acetate. Then, anti‐tumor components acting on Tyrosine 367 Cysteine‐fibroblast growth factor receptor 4 were screened and identified from extracts of Ligusticum wallichii. Components from the extract were retained on the cell membrane chromatographic column. The retained fraction was directly eluted into high‐performance liquid chromatography with mass spectrometry system for separation and identification. Finally, Levistolide A was identified as an active component from Ligusticum wallichii extracts. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide‐formazan colorimetric assay revealed that Levistolide A inhibits proliferation of overexpressing the mutated receptor cells with dose‐dependent manner. Phosphorylation of fibroblast growth factor receptor 4 was also decrease under Levistolide A treatment. Flex dock simulation verified that Levistolide A could bind with the tyrosine kinase domain of fibroblast growth factor receptor 4. Therefore, Levistolide A screened by the cell membrane chromatography combined with high‐performance liquid chromatography and mass spectrometry can arrest cell growth. In conclusion, the two‐dimensional high‐performance liquid chromatography method can screen and identify potential anti‐tumor ingredients that specifically act on the tyrosine kinase domain of the mutated fibroblast growth factor receptor 4.     

Natural estrogens in dairy products: Determination of free and conjugated forms by ultra high performance liquid chromatography with tandem mass spectrometry

Natural estrogens are synthesized by mammals in different amounts depending on the developmental stage and pregnancy/lactation period, and they may pass into milk, where they are mostly present as glucuronated and sulfated forms. In modern dairy practices, about 75% of milk is produced from pregnant cows; therefore, the amount of hormones that may pass into milk could be of concern. While estrogen determination in milk has been investigated in depth, the individual determination of estrogens and their conjugated forms in dairy products has not been fully addressed. The aim of this work was to develop and assess a sensitive method, using the peculiar retention properties of graphitized carbon black, to extract natural free estrogens and their major conjugated metabolites, without any enzymatic cleavage, from yogurt, cheese, and butter. The free and conjugated estrogens were eluted in two distinct fractions from the solid‐phase extraction cartridge and analyzed separately by ultra high performance liquid chromatography coupled to tandem mass spectrometry. Recoveries were higher than 80% for all the three sample typologies. The highest matrix effects were observed for butter, which was richest in lipid content, but was below 30%. A survey on some commercial dairy products suggests that production processes decreased estrogen content.     

Suitability of dispersive liquid–liquid microextraction for the in situ silylation of chlorophenols in water samples before gas chromatography with mass spectrometry

Trace analysis of chlorophenols in water was performed by simultaneous silylation and dispersive liquid–liquid microextraction followed by gas chromatography with mass spectrometry. Dispersive liquid–liquid microextraction was carried out using an organic solvent lighter than water (n‐hexane). The effect of different silylating reagents on the method efficiency was investigated. The influence of derivatization reagent volume, presence of catalyst and derivatization/extraction time on the yield of the derivatization reaction was studied. Different parameters affecting extraction efficiency such as kind and volume of extraction and disperser solvents, pH of the sample and addition of salt were also investigated and optimized. Under the optimum conditions, the calibration graphs were linear in the range of 0.05–100 ng/mL and the limit of detection was 0.01 ng/mL. The enrichment factors were 242, 351, and 363 for 4‐chlorophenol, 2,4‐dichlorophenol, and 2,4,6‐trichlorophenol, respectively. The values of intra‐ and inter‐day relative standard deviations were in the range of 3.0–6.4 and 6.1–9.9%, respectively. The applicability of the method was investigated by analyzing water and wastewater samples.