High resolution mass spectrometry based profiling of diet-related deoxyribonucleic acid adducts

Exposure of DNA to endo- and exogenous DNA binding chemicals can result in the formation of DNA adducts and is believed to be the first step in chemically induced carcinogenesis. DNA adductomics is a relatively new field of research which studies the formation of known and unknown DNA adducts in DNA due to exposure to genotoxic chemicals. In this study, a new UHPLC-HRMS(/MS)-based DNA adduct detection method was developed and validated. Four targeted DNA adducts, which all have been linked to dietary genotoxicity, were included in the described method; O⁶-methylguanine (O⁶-MeG), O⁶-carboxymethylguanine (O⁶-CMG), pyrimidopurinone (M1G) and methylhydroxypropanoguanine (CroG). As a supplementary tool for DNA adductomics, a DNA adduct database, which currently contains 123 different diet-related DNA adducts, was constructed. By means of the newly developed method and database, all 4 targeted DNA adducts and 32 untargeted DNA adducts could be detected in different DNA samples. The obtained results clearly demonstrate the merit of the described method for both targeted and untargeted DNA adduct detection in vitro and in vivo, whilst the diet-related DNA adduct database can distinctly facilitate data interpretation.     

高效液相色谱-同位素稀释质谱法定量分析人生长激素

建立了人生长激素( hGH)纯化分析和高效液相色谱( HPLC)与同位素稀释质谱( IDMS)联用高准确度绝对定量方法。采用快速蛋白液相色谱来分离纯化hGH,以傅里叶变换离子回旋共振质谱仪( FTICR-MS)准确地测定蛋白质的分子质量。纯化的hGH经酸水解后,采用KINETEX C18色谱柱为分析柱,以水(含0.1%三氟乙酸)和乙腈为流动相,等梯度分离,流速0.2 mL/min,温度40℃,采用电喷雾正离子模式进行电离,选择多反应监测模式进行检测,内标法定量分析。结果表明,FTICR-MS所测得的分子量实测值与理论值仅相差0.31 Da,脯氨酸、缬氨酸和苯丙氨酸在液相条件下5 min内达到基线分离,在最优条件下,hGH含量测定结果为186.80 mg/g,相对标准偏差为0.52%。利用本方法参加国际比对,比对结果与参考值等效一致。本方法具有简易、实用的特点,并且准确可靠,可作为hGH纯品标准物质的定值方法,为hGH的日常检测提供参考。     

High‐performance liquid chromatography separation of unsaturated organic compounds by a monolithic silica column embedded with silver nanoparticles

The optimization of a porous structure to ensure good separation performances is always a significant issue in high‐performance liquid chromatography column design. Recently we reported the homogeneous embedment of Ag nanoparticles in periodic mesoporous silica monolith and the application of such Ag nanoparticles embedded silica monolith for the high‐performance liquid chromatography separation of polyaromatic hydrocarbons. However, the separation performance remains to be improved and the retention mechanism as compared with the Ag ion high‐performance liquid chromatography technique still needs to be clarified. In this research, Ag nanoparticles were introduced into a macro/mesoporous silica monolith with optimized pore parameters for high‐performance liquid chromatography separations. Baseline separation of benzene, naphthalene, anthracene, and pyrene was achieved with the theoretical plate number for analyte naphthalene as 36 000 m^?1. Its separation function was further extended to cis/trans isomers of aromatic compounds where cis/trans stilbenes were chosen as a benchmark. Good separation of cis/trans‐stilbene with separation factor as 7 and theoretical plate number as 76 000 m^?1 for cis‐stilbene was obtained. The trans isomer, however, is retained more strongly, which contradicts the long‐ established retention rule of Ag ion chromatography. Such behavior of Ag nanoparticles embedded in a silica column can be attributed to the differences in the molecular geometric configuration of cis/trans stilbenes.     

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.     

Determination of five sesquiterpenoids in Xingnaojing injection by quantitative analysis of multiple components with a single marker

A quantitative analysis of multiple components with a single‐marker method was established for the simultaneous determination of five sesqutiterpenoids in Xingnaojing injection. This method was established with Xingnaojing injection determined by high‐performance liquid chromatography coupled with diode array detection. The durability and system suitability of the established method were evaluated, and the reliable relative correction factors were obtained with curdione selected as an internal reference. The contents of the five components in all Xingnaojing injections were determined by external standard method and the contents of curcumenone, curcumenol, curzerenone, and germacrone were also calculated with the obtained relative correction factors. Then, relative error was investigated to estimate the difference of the two methods. As a result, the established new method possesses good adaptability, and there is no significant difference between the two methods, except for the content of curzerenone in eight samples. To put the established method into practice, the limits of quantitation of the established method of the five components were proposed and defined. Thus, the developed methodology can also be utilized to the quality evaluation of Xingnaojing injection, in spite of the difference found in the content of curzerenone between the external standard method and the newly established method.     

Development and validation of polymerized high internal phase emulsion monoliths coupled with HPLC and fluorescence detection for the determination of trace tetracycline antibiotics in environmental water samples

A polymerized high internal phase emulsion monolith was used as a novel sorbent for solid‐phase extraction coupled with high‐performance liquid chromatography and fluorescence detection for the determination of oxytetracycline, tetracycline, doxycycline, and chlorotetracycline in environmental water samples. The polymerized high internal phase emulsion monolithic column was prepared by the in situ polymerization of the continuous phase of a high internal phase emulsion containing glycidyl methacrylate, styrene, and divinylbenzene in pipette tips, and then functionalized with iminodiacetic acid. The resulting monolith exhibited highly interconnected porosity and large surface areas, making it an excellent candidate as an solid‐phase extraction sorbent for the enrichment of trace tetracycline antibiotics. Several factors affecting the extraction performance of polymerized high internal phase emulsion monoliths, including the pH of sample solution, the eluting solvents, the sample loading flow rate and volume, were investigated, respectively. Under the optimized conditions, the mean recoveries of oxytetracycline, tetracycline, doxycycline, and chlorotetracycline spiked in pond and farm wastewater samples ranged from 78.1 to 119.3% with relative standard deviation less than 15%. The detection limits (S/N = 3) of the proposed method were in the range of 51–137 pg/mL. This study demonstrated that the monolithic polymerized high internal phase emulsion would be promising solid‐phase extraction sorbents in the extraction and proconcentration of trace analytes from complex samples.     

Solid‐phase extraction based on a molecularly imprinted polymer for the selective determination of four benzophenones in tap and river water

This work reports the preparation of molecularly imprinted polymer particles for the selective extraction and determination of four benzophenones from aqueous media. The polymer was prepared by using 4‐vinylpridine as functional monomer, ethylene glycol dimethacrylate as cross‐linker, acetonitrile as porogenic solvent and 2,2’,4,4’‐tetrehydroxybenzophenone as template. Good specific adsorption capacity (Q_max = 27.90 μmol/g) for 2,2’,4,4’‐tetrehydroxybenzophenone was obtained in the sorption experiment and good class selectivity for 2,2’,4,4’‐tetrehydroxybenzophenone, 2,4‐dihydroxybenzophenone, 2,2’‐dihydroxy‐4‐methoxybenzophenone, 2,2’‐dehydroxy‐4,4’‐dimethoxybenzophenone was demonstrated by the chromatographic evaluation experiment. Factors affecting the extraction efficiency of the molecularly imprinted solid‐phase extraction procedure were investigated systematically. An accurate and sensitive analytical method based on the molecularly imprinted solid‐phase extraction coupled with high‐performance liquid chromatography and diode array detection has been successfully developed for the simultaneous determination of four benzophenones from tap water and river water with method detection limits of 0.25–0.72 ng/mL. The recoveries of benzophenones for water samples at two spiking levels (500 and 5000 ng/mL for each benzophenone) were in the range of 86.9–103.3% with relative standard deviations (n = 3) below 9.2%.     

Pushing the speed limit in enantioselective supercritical fluid chromatography

Chromatographic enantioseparations on the order of a few seconds can be achieved by supercritical fluid chromatography using short columns packed with chiral stationary phases. The evolution of ‘world record’ speeds for the chromatographic separation of enantiomers has steadily dropped from an industry standard of 20–40 min just two decades ago, to a current ability to perform many enantioseparations in well under a minute. Improvements in instrument and column technologies enabled this revolution, but the ability to predict optimal separation time from an initial method development screening assay using the t_{min cc} predictor greatly simplifies the development and optimization of high‐speed chiral chromatographic separations. In this study, we illustrate how the use of this simple tool in combination with the workhorse technique of supercritical fluid chromatography on customized short chiral columns (1–2 cm length) allows us to achieve ultrafast enantioseparations of pharmaceutically relevant compounds on the 5–20 s scale, bringing the technique of high‐throughput enantiopurity analysis out of the specialist realm and into the laboratories of most researchers.     

Determination of phthalate esters from environmental water samples by micro‐solid‐phase extraction using TiO2 nanotube arrays before high‐performance liquid chromatography

We describe a highly sensitive micro‐solid‐phase extraction method for the pre‐concentration of six phthalate esters utilizing a TiO₂ nanotube array coupled to high‐performance liquid chromatography with a variable‐wavelength ultraviolet visible detector. The selected phthalate esters included dimethyl phthalate, diethyl phthalate, dibutyl phthalate, butyl benzyl phthalate, bis(2‐ethylhexyl)phthalate and dioctyl phthalate. The factors that would affect the enrichment, such as desorption solvent, sample pH, salting‐out effect, extraction time and desorption time, were optimized. Under the optimum conditions, the linear range of the proposed method was 0.3–200 μg/L. The limits of detection were 0.04–0.2 μg/L (S/N = 3). The proposed method was successfully applied to the determination of six phthalate esters in water samples and satisfied spiked recoveries were achieved. These results indicated that the proposed method was appropriate for the determination of trace phthalate esters in environmental water samples.     

Ionic liquid and aqueous two‐phase extraction based on salting‐out coupled with high‐performance liquid chromatography for the determination of seven rare ginsenosides in Xue‐Sai‐Tong injection

A method of ionic liquid salt aqueous two‐phase extraction coupled with high‐performance liquid chromatography has been developed for the analysis of seven rare ginsenosides including Rg₆, F₄, 20(S)‐Rg₃, 20(R)‐Rg₃, Rk₃, Rk₁, and Rg₅ in Xue‐Sai‐Tong injection. The injection was mixed with ionic liquid 1‐butyl‐3‐methylimidazolium bromide aqueous solution, and a mixture was obtained. With the addition of sodium dodecyl sulfate and dipotassium phosphate into the mixture, the aqueous two‐phase mixture was formed after ultrasonic treatment and centrifuged. Rare ginsenosides were extracted into the upper phase. To obtain a high extraction factors, various influences were considered systematically, such as the volume of ionic liquid, the category and amount of salts, the amount of sodium dodecyl sulfate, the pH value of system, and the time of ultrasonic treatment. Under the optimal condition, rare ginsenosides in Xue‐Sai‐Tong injection were enriched and detected, the recoveries of seven rare ginsenosides ranged from 90.05 to 112.55%, while relative standard deviations were lower than 2.50%. The developed method was reliable, rapid and sensitive for the determination of seven rare ginsenosides in the injections.