高效液相色谱串联质谱法快速测定烟草中5种甾醇的研究

本研究基于自主设计的集萃取、过滤和转移功能为一体的样品萃取瓶,建立了高效液相色谱串联质谱(LC-MS/MS)快速测定烟草中麦角甾醇、胆甾醇、菜油甾醇、豆甾醇和β-谷甾醇的方法。样品在萃取瓶中经水解、皂化和萃取,经LC-MS/MS进行分析检测。结果表明:自主设计的样品萃取瓶可提高前处理效率,5种甾醇在0.1～150 ng·mL~(-1)内均具有良好的线性关系(线性相关系数0.999);检出限在0.015～0.075ng·mL~(-1);平均回收率在93.4%～103.8%之间,日内精密度和日间精密度均小于3.5%,具有较好的重复性。该方法能够满足烟草中5种甾醇快速、准确测定。     

甾醇、甾烷醇柱前衍生高效液相荧光分析

采用荧光衍生化试剂2-(9-咔唑)乙酰氯,对天然产物谷甾醇、豆甾醇、菜油甾醇及谷甾烷醇的柱前衍生化条件,包括催化剂的种类、反应温度、衍生化时间等进行了考察,结果表明催化剂选用三乙胺,反应温度80℃,时间20 min,衍生产物具有恒定的最大检测响应值.利用高效液相色谱对衍生产物进行分析,并对衍生物的分离进行了优化选择,达到了较好的基线分离,检出限为12.6～29.5 nmol/L.方法应用于血清样品的测定,谷甾醇、豆甾醇、菜油甾醇和谷甾烷醇的标准加入回收率分别为101.9%、102.4%、101.0%和103.2%.     

混合植物甾醇中豆甾醇和β-谷甾醇的高效液相色谱分析

应用高效液相色谱对豆甾醇、β_谷甾醇和重结晶法分离精制的混合甾醇进行了分析测定 ;实验采用的色谱柱为HypersilODS反相柱(4.6×150mm ,5μm) ,流动相为甲醇 ,紫外检测波长为210nm ,恒溶剂洗脱 ,混合植物甾醇在10min内得到了很好的分离 ;实验结果表明 ,采用液相色谱法能够快速准确地测定混合甾醇中豆甾醇和 β_谷甾醇的含量。     

甾醇、甾烷醇柱前衍生高效液相荧光分析

采用荧光衍生化试剂2-（9-咔唑）乙酰氯，对天然产物谷甾醇、豆甾醇、菜油甾醇及谷甾烷醇的柱前衍生化条件，包括催化剂的种类、反应温度、衍生化时间等进行了考察，结果表明：催化剂选用三乙胺，反应温度80℃，时间20min，衍生产物具有恒定的最大检测响应值。利用高效液相色谱对衍生产物进行分析，并对衍生物的分离进行了优化选择，达到了较好的基线分离，检出限为12．6～29．5nmol／L。方法应用于血清样品的测定，谷甾醇、豆甾醇、菜油甾醇和谷甾烷醇的标准加入回收率分别为101．9％、102．4％、101．0％和103．2％。     

溶剂萃取-气相色谱/质谱法分析烟草中的主要甾醇

烟草中的甾醇类物质主要有胆甾醇、菜油甾醇、豆甾醇和β-谷甾醇等,这些甾醇的结构中都含有羟基（结构式见图1）,热解时其母体的多环结构可形成稠环芳烃,因此烟草中的甾醇是一种潜在的影响人体健康的物质,故对甾醇种类和含量进行分析对卷烟的配方研究具有参考价值。     

大豆甾醇及其异构体的分离与鉴定

筛选出适合分离大豆甾醇的最佳反相色谱柱W aters symm etry C18(4.6mm×250mm)和流动相(乙腈-异丙醇,95∶5,V/V),考察了流动相配比和柱温对大豆甾醇分离的影响。从大豆甾醇产品中基线分离出豆甾醇、β-谷甾醇和菜油甾醇及菜籽甾醇这4种主要甾醇,还通过对分离出的其它多个未知峰馏分收集制备后作GC/MS分析,鉴定为γ-谷甾醇、24-β-菜油甾醇、表菜籽甾醇和豆甾醇异构体。在优化的色谱条件下,利用HPLC测定了大豆油和脱臭馏出物及大豆甾醇产品的部分样品中各甾醇组分的含量,豆甾醇和β-谷甾醇的检出限为14.4 ng和10.6 ng,HPLC测定大豆油样品中这2种甾醇的平均回收率为97.3%和96.9%。     

烟草中植物甾醇的GC-MS分析

建立了一种GC-MS同时测定烟草中胆甾醇、菜油甾醇、豆甾醇及β-谷甾醇的分析方法。结果表明:胆甾醇、菜油甾醇、豆甾醇及β-谷甾醇回收率在95.94～102.41%之间,RSD<6.01%。在所测定的烟草样品中,豆甾醇的含量较高。所检测的烟草样品中,均未检测出糖苷态胆甾醇。     

反相高效液相色谱法分析禾谷丝核菌菌丝中麦角甾醇和羊毛甾醇的含量

采用反相高效液相色谱法,建立了检测禾谷丝核菌中麦角甾醇和羊毛甾醇含量的方法.样品经过破碎、离心、皂化后定量测定.采用DiamonsilTMC18色谱柱(250 mm×4.6 mm i.d, 5 μm i.d.),流动相为100%甲醇,流速为1 mL/min,紫外检测波长为210 nm,保留时间定性,外标法定量.麦角甾醇和羊毛甾醇的保留时间分别为21 min和27min.麦角甾醇在0.125～12.5 mg/L、羊毛甾醇在0.1～10.0 mg/L 范围内,两者峰面积与浓度均呈线性关系,相关系数分别为0.9993和0.9983.添加回收率实验表明,2种甾醇的平均添加回收率为91.8%～99.7%;相对标准偏差为2.8%～7.8%;麦角甾醇和羊毛甾醇的最小检出量分别为1.0和0.8 ng,最低检出浓度分别为0.05和0.04 mg/L.本方法准确、简便、重现性好.     

β-谷甾醇、豆甾醇与DPPC相互作用的单分子膜研究

植物甾醇对生物膜具有重要的调节作用,甾醇尾部的饱和程度对生物膜性质的影响一直是个热点问题.通过LB技术和原子力显微镜分别研究了尾部是单键的β-谷甾醇(β-sito)和尾部是双键的豆甾醇(stig)与二棕榈酰磷脂酰胆碱(DPPC)的相互作用,深入分析了β-谷甾醇、豆甾醇对DPPC单分子膜液态扩张-液态聚集(LE-LC)相变过程的影响.实验结果表明:β-谷甾醇、豆甾醇添加到DPPC单分子膜中,膜的压缩性以及分子的排列都发生了较大变化.当植物甾醇含量Xsterols=0.8时,β-谷甾醇、豆甾醇与DPPC相互作用具有相同的规律,过量分子面积和过量吉布斯自由能均为负值,说明分子间的吸引力比排斥力更强;在低浓度下,Xsterols=0.2,0.4时,两种系统的过量分子面积和过量吉布斯自由能呈现出明显的区别,尾部是单键的β-谷甾醇和DPPC混合单分子膜的排斥力更强烈,而尾部是双键的豆甾醇和DPPC混合单分子膜的吸引力更强烈,说明尾部是双键的豆甾醇比尾部是单键的β-谷甾醇更容易和DPPC发生凝聚,AFM数据进一步证实了这些结果.     

超声萃取-高效液相色谱-串联质谱法测定大气颗粒物中甾醇类化合物

采用超声萃取与液相色谱-串联质谱联用,建立了快速测定大气颗粒物中甾醇类化合物的方法.甾醇类化合物用甲醇超声萃取,浓缩后使用液相色谱-串联质谱分析.采用Waters公司Atlantis C18色谱柱(100mm× 2.1 mm,3μm),以乙腈和水混合流动相梯度洗脱,实现了胆甾醇、豆甾醇、菜油甾醇及β-谷甾醇的分离.并在APCI-MS/MS MRM模式下定量检测.在选取的实验条件下,方法回收率在80.3％～97.7％之间,检出限0.015 ng/m3,相对标准偏差小于15％,日内及日间测定精密度小于20％.本方法具有较好的准确性及精密度,实际样品的测试结果表明,方法可以满足大气颗粒物中甾醇类化合物的定量分析要求.     

Total Triterpenoid Extraction from Inonotus Obliquus Using Ionic Liquids and Separation of Potential Lactate Dehydrogenase Inhibitors via Ultrafiltration High-Speed Countercurrent Chromatography

Extracts of the fungus Inonotus obliquus exhibit cytotoxic properties against different cancers; hence, this fungal species has been extensively studied. This study aimed to extract total triterpenoids from Inonotus obliquus using ionic liquids (ILs) and separate potential lactate dehydrogenase (LDH) inhibitors via ultrafiltration (UF)-high-speed countercurrent chromatography (HSCCC). Total triterpenoids from Inonotus obliquus were extracted by performing a single-factor experiment and employing a central composite design via ultrasonic-assisted extraction (UAE) and heat-assisted extraction (HAE). The extract was composed of 1-butyl-3-methylimidazolium bromide as the IL and methanol as the dispersant. Ultrafiltration-liquid chromatography (UF-LC) was used to rapidly scan the LDH inhibitors and betulin and lanosterol were identified as potential inhibitors. To obtain these target compounds, betulin and lanosterol with the purities of 95.9% and 97.8% were isolated from HSCCC within 120 min. Their structures were identified using several techniques, among which IL-HAE was fast and effective. This study reports the extraction of triterpenoids from Inonotus obliquus by IL for the first time. Collectively, the findings demonstrate that UF-LC is an effective tool for screening potential LDH inhibitors from crude extracts of I. obliquus and may help to identify bioactive substances against myocardial infarction, whereas high-purity compounds can be separated via UF-HSCCC.     

Supercritical CO2 Extraction of Triterpenoids from Chaga Sterile Conk of Inonotus obliquus

Triterpenoids are among the bioactive components of Chaga, the sterile conk of the medicinal fungus Inonotus obliquus. Supercritical fluid extraction of Chaga triterpenoids was carried out with supercritical CO₂, while a modified Folch method was used as a comparison. Three temperature-pressure combinations were tested varying between 314–324 K (40–50 °C) and 281–350 bars, using time- and volume-limited extractions. Six triterpenoids were identified with GC-MS and quantified with GC-FID: ergosterol, lanosterol, β-sitosterol, stigmastanol, betulin, and inotodiol. The Folch extraction resulted in recovery of trametenolic acid, which was not extracted by supercritical CO₂. Inotodiol was the major triterpenoid of all the extracts, with a yield of 87–101 mg/100 g and 139 mg/100 g, for SFEs and the Folch method, respectively. The contents of other major triterpenoids, lanosterol and ergosterol, varied in the ranges 59–63 mg/100 g and 17–18 mg/100 g by SFE, respectively. With the Folch method, the yields were 81 mg/100 g and 40 mg/100 g, respectively. The highest recovery of triterpenoids with SFE in relation to Folch was 56% and it was obtained at 324 K (50 °C) and 350 bar, regardless of extraction time or volume of CO₂. The recoveries of lanosterol and stigmastanol were unaffected by SFE conditions. Despite the lower yield, SFE showed several advantages including shorter extraction time and less impact on the environment. This work could be a starting point for further studies on green extraction methods of bioactive triterpenoids from Chaga.     

Simultaneous extraction and determination of free and conjugated phytosterols in tobacco

Acid hydrolysis and alkaline saponification were incorporated into a microwave‐assisted extraction process for the simultaneous extraction of free and conjugated phytosterols from tobacco. The crude extract of the microwave‐assisted extraction was purified by C₁₈ solid‐phase extraction and then determined by high‐performance liquid chromatography. Phytosterols of cholesterol, ergosterol, stigmasterol, campesterol, and β‐sitosterol were determined by chromatographic quantification. The multiple parameters of microwave‐assisted extraction were optimized by a uniform design method. The optimal ratio of extraction ethanol solvent to tobacco mass was 30 mL/g. The microwave‐assisted extraction acid hydrolysis was carried out in sulfuric acid medium by heating for 10 min at 55°C. The microwave‐assisted extraction alkaline saponification was performed after adding excessive sodium hydroxide by heating another 10 min. The repeatability of the proposed method was acceptable with recoveries from 69.68 to 88.17% for the phytosterols. Five target phytosterols were all found in the tobacco samples, and the contents were significantly different in samples from different producing areas.     

不同固醇与DPPC二元体系的液态有序相

应用同步辐射X射线衍射和差示扫描量热法研究了由不同结构的固醇(胆固醇、脱氢胆固醇、豆固醇、谷固醇、麦角固醇以及固醇核)和二棕榈酰磷脂酰胆碱(DPPC)二元体系形成的液态有序相. 研究表明, 胆固醇比植物固醇(豆固醇和谷固醇)和真菌固醇(麦角固醇)能更有效地与DPPC形成液态有序相(Lo); 有胆固醇或者脱氢胆固醇参与的液态有序相能够在较宽的温度范围内保持稳定, 而由植物固醇和真菌固醇参与的液态有序相对温度有较强的依赖性, 在DPPC主相变温度附近有明显的热致相变过程, 因此这一液态有序相应该进一步区分为Loβ和Loα相. 研究结果有助于阐明固醇尾链在液态有序相以及脂筏中的作用, 也有助于理解在进化过程中动物细胞膜为何选择胆固醇作为主要固醇.     

Development of a fast sample treatment for the analysis of free and bonded sterols in human serum by LC‐MS

The analysis of sterols in biological fluids allows the clinical study of cholesterol related diseases. This research is focused on reducing the sample processing time of the determination of free and bonded sterols in human serum. Ten sterols were studied: cholesterol precursors (desmosterol, lanosterol, and cholestanol); phytosterols (stigmasterol, campesterol, sitosterol, and sitostanol) and oxysterols (7‐α‐hydroxy‐4‐cholesten‐3‐one, 24‐hydroxycholesterol, and 27‐hydroxycholesterol). Ultrasound assistance was used to diminish the reaction time during the alkaline hydrolysis for determining total sterols. Different retention mechanisms of solid‐phase extraction were compared, two reversed‐phase sorbents DSC‐18 and polymeric Oasis‐HLB and a novel zirconia‐coated silica phase. DSC‐18 and zirconia‐coated silica were the most suitable sorbents to analyze these metabolites. The resulting extracts were analyzed by liquid chromatography coupled to mass spectrometry. The analytical parameters were determined and better values were observed with DSC‐18 cartridges for most sterols. LOQ were in the low ng/mL level. Recoveries were in the range 85–99%. Average intermediate precision was 15%. Accuracy for both cartridges was more than 92%. Zirconia‐coated silica showed better performance for the oxysterols, with recoveries around 90%. The procedure allows the determination of free and bonded sterol precursors, phytosterols, and oxysterols in human serum.     

Extraction of ergosterol from peaty soils and determination by high performance liquid chromatography

The ergosterol content of soil can be used as an indicator of fungal activity. A method has been developed for the extraction and determination of ergosterol in organic soils, as part of a study to assess the correlation between fungal activity and the sequestration of metal pollutants. The moisture content of the soil affected the extraction process. Four consecutive extractions with methanol removed >95% of the ergosterol that can be obtained from the fresh sample (63% moisture) by exhaustive extraction. By freeze drying the soils prior to extraction (a) up to 35% more ergosterol was extracted after a single extraction, (b) >90% of the recoverable ergosterol was collected in two extractions and (c) the repeatability of the extraction was improved. Storage of soil extracts in the absence of light prevents degradation of ergosterol. A previously reported method for determination of ergosterol by HPLC has been improved by modification of the eluant composition. With 46% methanol/46% acetonitrile/8% dichloromethane, ergosterol was eluted with good resolution approximately 8 min after injection of 20 mul of the extract. The detection limit of the HPLC method was 0.5 mug/ml ergosterol, equivalent to 0.06 mug/g in 25 g fresh soil. Changes in ergosterol contents of peaty soil treated with fungicide, and in samples of the peaty podzol and a humus iron podzol in the vicinity of fungal fruiting bodies, have been determined.     

Liquid Ordered Phase of Binary Mixtures Containing Dipalmitoylphosphatidylcholine and Sterols

The effect of cholesterol, desmosterol, stigmasterol, sitosterol, ergosterol, and androsterol on the phase behavior of aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) was studied to understand the role of the side chain in the formation of ordered phases of the type observed in membrane rafts. Thermotropic changes in the structure of mixed dispersions and transition enthalpies were examined by synchrotron X-ray diffraction (XRD) and differential scanning calorimetry (DSC). The observations indicated that cholesterol was more efficient than phytosterols (stigmasterol and sitosterol) or ergosterol in its interaction with DPPC to form the liquid ordered phase (L_o). The L_o induced by cholesterol or desmosterol was stable over a wide temperature range, whereas, the liquid ordered phase containing phytosterols or ergosterol was profoundly dependent on temperature, which should be distinguished as L_oβ and L_oα, representing the phases below and above the main transition temperature. The characteristics in forming ordered structures of cholesterol and other sterols imply that the evolution may have selected cholesterol as the most efficient sterol for animals to form rafts in their cell membranes.     

Separation and determination of diversiform phytosterols in food materials using supercritical carbon dioxide extraction and ultraperformance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry

This paper presents at first time that the ultra-performance™ liquid chromatographic atmospheric pressure chemical ionization mass spectrometer (UPLC-APCI-MS) was used as an efficient method for the identification and quantification of diversiform phytosterols in food materials. The sample preparation consisted of extraction by supercritical carbon dioxide fluid extraction (SCE) and saponification by refluxing with ethanolic KOH, and then the non-saponificable fraction was extracted with petroleum ether. This fraction was subjected to solid phase extraction (SPE) on silica gel cartridge and then the sterols were eluted with hexane-ethyl acetate. Sterols were separated on an Acquity UPLC™ BEH C18 column (100 mm × 1.0 mm, 1.7 μm particle size) with a gradient of methanol/water (1% acetonitrile) at a flow of 0.1 mL min⁻¹. The determination was performed in selective ion monitoring mode. The quality parameter of the developed method was established using 6-ketocholestanol as internal standard. Limits of quantification (LOQ) were 0.1754, 0.0341, 0.0500, 0.0205, 0.0225, 0.3674, 0.0241, 0.0272, 0.0076 μg L⁻¹ and 0.1525 μg mL⁻¹ for 6-ketocholestanol, desmosterol, ergosterol, cholesterol, lanosterol, cholestanol, campesterol, stigmasterol, β-sitosterol, and stigmastanol, respectively. The intra- and inter-day determination precision for the 10 phytosterols were less than 5 and 6% in relative standard deviations, and their recoveries were located in the range of 94-107%. The developed approach has been applied successfully for efficient determination of diversiform phytosterols in food materials, including corn, sesame, oat and peanut.     

Liquid chromatography-UV determination and liquid chromatography-atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soybean oil.

A narrow-bore HPLC-UV method was developed for the analysis of two of the more abundant naturally occurring phytosterols in vegetable oils: sitosterol and stigmasterol. The method enabled detection of the compounds at a concentration of 0.42 microg/ml and quantitation at concentrations of 0.52 and 0.54 microg/ml for sitosterol and stigmasterol, respectively. An excellent linearity was determined over two orders of concentration magnitude (r2 0.999-1.000) and verified by applying the Mandel fitting test (p>0.099) and the lack-of-fit test (p>0.057) performed at the 95% confidence level. A good intra-day precision ranging from 0.15 to 1.16% was calculated at two concentration levels (2 and 100 microg/ml). The inter-day reproducibility was verified on 3 different days by performing an homoscedasticity test and analysis of variance. A solid-phase extraction method was developed on silica cartridges for the isolation of phytosterols from soybean oil providing recovery values of 101+/-9 and 106+/-7% for sitosterol and stigmasterol, respectively. Good accuracy of the method was statistically demonstrated since no matrix effect was found for both the analytes. The developed method was applied to the quantitative assay of phytosterols in a soybean oil sample (61+/-5 mg/100 g of stigmasterol and 118+/-4 mg/100 g sitosterol). The HPLC-atmospheric pressure chemical ionization MS technique enabled the identification of stigmasterol, sitosterol and campesterol in the oil sample.