高效液相色谱法测定人和小鼠粪便中短链脂肪酸

目的建立高效液相色谱测定人和小鼠粪便中短链脂肪酸的方法。方法样品进行衍生化处理,采用高效液相色谱法测定乙酸、丙酸、丁酸以及戊酸。YMC-Pack FA型色谱柱(250 mm×6.0 mm,5μm),流动相甲醇–水(60∶40,0.1%三氟乙酸调节p H值至4.5),检测波长400 nm,体积流量1.0 m L/min,柱温50℃,进样量20μL。结果乙酸、丙酸、丁酸、戊酸分别在0.300~6.005 mg/L(r=0.999 2)、0.370~7.408 mg/L(r=0.998 5)、0.441~8.811 mg/L(r=0.997 3)、0.511~10.210 mg/L(r=0.997 9)线性关系良好;平均加样回收率分别为93.27%、96.40%、95.67%、95.43%,RSD值分别为4.62%、5.42%、3.64%、3.92%。结论方法操作简单,精密度、稳定性、重复性良好,可用于测定人、小鼠粪便中短链脂肪酸。     

柱前衍生气质联用法测定罂粟籽油中的脂肪酸组分

建立了柱前衍生气相色谱-质谱法对罂粟籽油中的20种脂肪酸组分进行定性定量分析。样品经氢氧化钾甲醇溶液皂化、三氟化硼甲醇溶液甲酯化、正庚烷萃取后,使用WM-CN100色谱柱进行色谱分离,以全扫描(Scan)和选择离子扫描(SIM)模式进行测定。其中,36种脂肪酸甲酯均有较好的线性关系,相关系数(R2)≥0.9970;检测限均小于40 ng/ml;系统精密度RSD≤8.9%;样品测定重复性RSD(SD)≤9.6%;四种较大组分平均回收率为98.4%~108.5%,RSD≤9.1%。3批市售罂粟籽油中存在主要脂肪酸组分4种、微量脂肪酸组分13种、反式脂肪酸2种及1种未知组分。其中,反式脂肪酸总量为2.59e-02~3.08e-02g/100g。本研究建立的检测方法灵敏度高、专属性好、结果准确,能对罂粟籽油等植物油中的主要及微量脂肪酸组分进行检测。     

气相色谱-质谱联用法分析肠炎小鼠短链脂肪酸代谢

目的 建立气相色谱-质谱联用（GC/MS）的短链脂肪酸（SCFAs）分析方法，分析肠炎小鼠SCFAs代谢情况。方法 盐酸饱和氯化钠溶液酸化生物样本，醋酸乙酯提取SCFAs后衍生化，建立GC/MS方法进行生物样本中SCFAs测定；考察盐酸溶液提取和盐酸饱和氯化钠溶液提取SCFAs总离子流图谱；考察SCFAs混合物衍生化前后的稳定性，粪便、血清、小肠、结肠、肝脏组织中SCFAs测定方法的线性、精密度及准确度、提取回收率和基质效应。运用建立的方法对正常及硫酸葡聚糖（DSS）慢性肠炎小鼠血、肝、小肠、结肠、结肠内容物以及盲肠内容物中发挥重要作用的SCFAs进行测定。结果 用盐酸饱和氯化钠溶液进行SCFAs提取后，建立的GC/MS法可同时测定甲酸、乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸、异己酸、己酸、乳酸、琥珀酸共11种SCFAs；方法学考证显示，该方法具有较好的准确度、精密度以及提取回收率；除乙酸外，其余SCFAs基质效应均较好，不能完全除去乙酸的基质效应，但其RSD均小于3.8%，说明基质的影响效应相对稳定，对结果准确性影响较小。与对照组比较，慢性肠炎小鼠结肠内容物SCFAs蓄积；盲肠内容物SCFAs整体下调，乳酸蓄积，提示肠炎小鼠SCFAs代谢紊乱。结论 建立了生物样品中SCFAs提取和检测方法，并应用于慢性肠炎小鼠SCFAs代谢分析，肠炎小鼠SCFAs代谢紊乱。     

鱼肝油中脂肪酸的气相色谱及气相色谱-质谱分析

目的 采用气相色谱法（GC）结合气相色谱-质谱（GC-MS）联用技术,对3种不同来源共26批鱼肝油样品中的脂肪酸成分进行测定。方法 通过氢氧化钾-甲醇碱催化法对鱼肝油样品进行甲基衍生化预处理,选用极性毛细管柱对样品中的衍生化产物脂肪酸甲酯进行分离,后经气相色谱仪-氢火焰离子化检测器（GC-FID）和气相色谱-质谱（GC-MS）联用仪进行分析检测。利用对照品定位法结合NIST谱库检索准确鉴定出棕榈酸、棕榈油酸、油酸、亚油酸、二十碳五烯酸（eicosapentaenoic acid,EPA）和二十二碳六烯酸（docosahexaenoic acid,DHA）等14种脂肪酸,并通过面积归一化法对这14种脂肪酸进行定量分析。结果 这14种脂肪酸在国内鱼肝油中的含量与模拟天然鱼肝油和进口鱼肝油样品中的含量存在较大差异。其中,EPA和DHA等脂肪酸在模拟天然鱼肝油和进口鱼肝油样品中的含量远高于其在国内鱼肝油样品中的含量,而亚油酸在国内鱼肝油样品中的含量却高达44%以上,10倍于其在另外2种鱼肝油样品中的含量。结论 不同来源的鱼肝油样品中的脂肪酸成分不尽相同,可根据脂肪酸的组成区别鱼肝油样品中是否添加天然鱼肝油成分。     

气-质联用法测定银杏叶片中α-亚麻酸的含量

目的建立一种用气相色谱/质谱联用(GC-MS)技术测定绿康银杏叶片中α-亚麻酸的含量,以期完善其质量标准.方法先将样品中紫苏籽油进行甲酯化后测定各脂肪酸的组成,并对总离子流图中各色谱峰相应的质谱图经人工解析图谱及计算机检索,确定其化学结构.色谱柱为FFAP 30 m×25 mm×0.25μm;柱前压为80 kPa;柱温为100～220℃;进样口温度为250℃.结果共鉴定出4种脂肪酸,分别为棕榈酸、油酸、亚油酸、α-亚麻酸,相对百分含量分别为7.094%,21.032%,17.588%,54.285%.并且α-亚麻酸在o.201 2～2.012 g·L-1范围内线性关系良好,r=0.999 2,平均回收率为99.01%,RSD为1.35%;成品中的平均含量为1.111 mg/片.结论此种测定方法稳定性、重现性好,并且紫苏籽油中α-亚麻酸的相对百分含量最高,可以作为绿康银杏叶片的一项定量质控指标.     

GC-MS法同时测定糖尿病肾病大鼠血浆中25种脂肪酸

目的建立柱前衍生化结合气-质联用法(gas chromatography-mass spectrometry,GC-MS)同时定量测定大鼠血浆中25种脂肪酸的分析方法,并应用于糖尿病肾病大鼠的测定。方法采用NaOH-CH_3OH和BF_3-CH_3OH两步衍生化对血浆样品进行处理,以高纯氦气为载气,Agilent DB-23(60 m×0. 25 mm,0. 15μm)毛细管柱为固定相,程序升温;选择离子监测定量测定25种脂肪酸。结果25种脂肪酸得到较好分离,且在各自测定范围内其质量浓度与响应值具有良好的线性关系(r>0. 995);批内及批间精密度(RSD)均小于15%;样品平均加样回收率在85. 17%～113. 3%内;血浆样品衍生化后-20℃放置24 h均稳定。结论该方法简便易操作,而且该衍生化产物稳定、线性范围宽,可用于糖尿病肾病大鼠血浆中脂肪酸的测定。     

鱼胶的化学成分及其对环磷酰胺所致贫血小鼠的改善作用研究

目的 研究鱼胶的化学成分及其对环磷酰胺所致贫血小鼠的改善作用。方法 采用硫酸苯酚法测定总糖含量,柱前衍生高效液相色谱法测定单糖组成,凯氏定氮法测定粗蛋白含量,氨基酸自动分析仪测定氨基酸组成,全自动脂肪测定仪测定粗脂肪含量,气相色谱质谱法测定脂肪酸组成,电感耦合等离子体质谱法测定矿物元素,采用胃蛋白酶辅助提取法提取获得了胶原蛋白,并对胶原蛋白含量、组成与结构进行了分析。采用腹腔注射环磷酰胺的方法建立小鼠贫血模型,观察对小鼠血细胞及脏器系数的影响。结果 总糖含量小于0.4%,单糖组成以葡萄糖、半乳糖为主；蛋白质含量在97.0～98.1%之间,含有17种氨基酸,包含7种人体必需氨基酸；脂肪酸含量在1.0～1.8%之间,脂肪酸组成以棕榈酸、硬脂酸、油酸为主；富含钙元素,且铁、锌、铜、锰、硒5种微量元素含量丰富；胶原蛋白含量在11.2~73.5%之间,氨基酸组成分析表明甘氨酸含量最高,在18.8～20.6%之间；在135 kDa左右有一条α-链、200 kDa左右有一条β-链、245 kDa左右有一条γ-链,紫外光谱、傅里叶变换红外光谱分析和圆二色谱结果均符合Ⅰ型胶原蛋白的特征。体内补血实验结果表明鱼胶可以促进模型小鼠胸腺指数、外周血红细胞、血红蛋白、血小板计数等的恢复。结论 首次对鱼胶进行了系统的化学成分研究,且鱼胶能促进环磷酰胺模型小鼠造血机能的恢复,为后续阐明鱼胶药效物质奠定基础。     

厌氧菌代谢产物中短链脂肪酸的气相色谱测定

厌氧菌依靠发酵取得赖以生存和繁殖的能量。在代谢过程中产生较多的挥发性和非挥发性短链脂肪酸产物,而不同种厌氧菌产生的脂肪酸种类和数量往往不同,因此气相色谱在厌氧菌的鉴定、分类和厌氧菌感染的诊断上具有一定的价值[1,2]. 文献[3,4,5]报道的测定厌氧菌代谢产物中非挥发性或挥发性短链脂肪酸的方法,多先将样品经繁琐、费时的衍生化成它的甲酯(尤其非挥发性酸,如     

减肥食品及保健食品中非法添加化学药物酚酞、盐酸西布曲明的探索性检测

目的:建立减肥食品及保健食品中非法添加化学药物酚酞、盐酸西布曲明的定性、定量检测方法。方法:采用薄层色谱法初筛,高效液相色谱-质谱联用法进行定性鉴别,高效液相色谱法同时进行含量测定。结果:薄层色谱鉴别斑点清晰,重复性好;高效液相色谱-质谱联用鉴别专属性强,灵敏度高;高效液相色谱含量测定方法准确可靠,酚酞、盐酸西布曲明浓度分别在2.9～175.3μg·ml^-1（r=0.999 8）和2.7～161.5μg·ml^-1（r=0.999 9）范围内与峰面积呈良好的线性关系,平均回收率分别为98.41%（RSD=1.61%）和100.83%（RSD=1.63%）。结论:本方法可有效监测减肥食品及保健食品中非法添加酚酞、盐酸西布曲明。     

柱前衍生化-HPLC法测定文冠果种仁油中脂肪酸含量

目的:建立文冠果种仁油中脂肪酸含量的HPLC 测定法.方法:以ω-溴苯乙酮为衍生化试剂,三乙醇胺为相转移催化剂,采用BDS C18色谱柱(250mm×4.6mm,5μm),检测波长235nm,以甲醇∶乙腈∶水(58∶27∶15)为流动相等度洗脱,柱温30℃,流速1.0mL·min-1,一次性分离五种脂肪酸,建立文冠果种仁油中脂肪酸含量的测定方法.结果:亚麻酸的线性范围为9.54～63.6μg,相关系数为0.9990,加样回收率为102.2%、RSD 为2.78%(n=9).结论:本方法重现性好,测量准确,可作为文冠果种仁油中脂肪酸含量测定的定量方法,用于文冠果种仁油的质量控制.     

液-质联用测定Nrf2野生型和基因敲除型小鼠肝脏中16种胆汁酸

目的:建立测定小鼠肝脏中16种胆汁酸浓度的LC-MS/MS法,并将测定结果应用于比较Nrf2野生型和基因敲除型小鼠胆汁酸代谢谱。方法:小鼠肝脏样品加入70%乙腈匀浆,用甲醇沉淀匀浆液中的蛋白并高速离心,取上清液置进样瓶, 用Xtimate^TM C₁₈柱分离,以甲醇-醋酸铵及甲酸水溶液为流动相进行梯度洗脱,流速0.25 ml·min^-1,进样5 μl分析。结果:16种胆汁酸在0.013~8.000 μmol·L^-1范围内,线性关系良好,定量下限浓度为0.013 μmol·L^-1,肝脏匀浆液加样回收率在94.9%~112.8%之间,日内、日间RSD均小于10.8%。结论:该方法准确可靠,灵敏度高,处理简便,适用于肝脏中胆汁酸浓度的测定,为生物样品中胆汁酸含量测定提供参考。     

Determination of free fatty acids in rabbit plasma by GC-MS after trimethylsilylation derivatization

In the present study, free fatty acids (FFAs, including palmiticacid, stearic acid, oleic acid, linoleic acid and arachidonicacid) in rabbit plasma were determined by gas chromatography-mass spectrometry (GC-MS) after trimethylsilylation derivatizationusing N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) – trimethylchlorosilane (TMCS) as derivatization reagent. The experimental conditions, including extraction and silylation reaction, were investigated. The method was experimentally validated. The linearity between fatty acids’ peak areas and their concentrations was obtained with the corelative coefficient (r²) all more than 0.999, and the recoveries were between 82% and 111%. The intra-day variations of FFAs’ in plasma samples at different concentrations were all less than 6%. FFA analysis results of 16 rabbit plasma samples showed that the method could be well applied in the determination of plasma samples in vivo. In contrast to the traditional method of FFA derivatization, the established trimethylsilylationmethod presented simplicity, high specificity, and completely free from the interference of the esterified fatty acid, such as triacylglyceride. The method could be applied for analyzing FFA profiles in the clinical laboratory or pharmacological research.     

LC-MS/MS法测定人粪便中匹诺塞林的浓度及在粪便排泄中的应用

目的建立LC-MS/MS测定人粪便中匹诺塞林浓度并将其应用于粪便排泄的方法。方法样品用乙腈提取处理,用LunaTMC8色谱柱,以乙腈-乙酸铵水为流动相,等度洗脱(65∶35),流速为0.25 mL.min-1,电喷雾电离源(ESI),用负离子模式检测,多反应离子监测(MRM)。结果匹诺塞林的线性范围为30～4000 ng.mL-1,定量下限为30 ng.mL-1,日间、日内准确度与精密度变异均<7%,回收率均>90%,不存在基质效应。48 h内粪便的累积排泄率为(1.9±1.4)%。结论本方法快速、灵敏、专属性强、重现性好,可用于人粪便中的匹诺塞林测定。     

高效液相色谱荧光检测法测定小鼠脑组织中氨基酸类神经递质(英文)

建立并优化高效液相色谱荧光检测法,灵敏有效地检测5种氨基酸类神经递质在小鼠脑组织中的浓度。以高丝氨酸(Hse)为内标,经过邻苯二甲醛柱前衍生化后,检测天门冬氨酸(Asp)、谷氨酸(Glu)、甘氨酸(Gly)、牛磺酸(Tau)和γ-氨基丁酸(GABA)。通过等度洗脱,5种氨基酸在24分钟内被精确分离。该方法在定量范围内线性良好,相关系数不低于0.9998,且检测限不超过10nmol/L。以相对标准偏差表示批内及批间重现性均小于或等于10.5%。该方法被成功应用于C57BL/6小鼠黑质和纹状体中氨基酸类神经递质浓度检测。     

Gas chromatography-mass spectrometry-based analytical strategies for fatty acid analysis in biological samples

Fatty acids play critical roles in biological systems. Imbalances in fatty acids are related to a variety of diseases, which makes the measurement of fatty acids in biological samples important. Many analytical strategies have been developed to investigate fatty acids in various biological samples. Due to the structural diversity of fatty acids, many factors need to be considered when developing analytical methods including extraction methods, derivatization methods, column selections, and internal standard selections. This review focused on gas chromatography-mass spectrometry (GC–MS)-based methods. We reviewed several commonly used fatty acid extraction approaches, including liquid–liquid extraction and solid-phase microextraction. Moreover, both acid and base derivatization methods and other specially designed methods were comprehensively reviewed, and their strengths and limitations were discussed. Having good separation efficiency is essential to building an accurate and reliable GC–MS platform for fatty acid analysis. We reviewed the separation performance of different columns and discussed the application of multidimensional GC for improving separations. The selection of internal standards was also discussed. In the final section, we introduced several biomedical studies that measured fatty acid levels in different sample matrices and provided hints on the relationships between fatty acid imbalances and diseases.     

LC/ESI/MS analysis of saturated and unsaturated fatty acids in rat intestinal epithelial cells

Reactive oxygen species (ROS) can mediate damage to cellular macromolecules and lipids. Lipid peroxidation is considered to be a major pathway by which ROS can cause tissue damage and alterations in cell membranes. Other factors affecting oxidative damage include the target molecules such as fatty acids, which are readily oxidized by ROS. Thus, lipid peroxidation may depend upon the cellular fatty acid composition. Analysis of saturated fatty acids that are present by liquid chromatography/mass spectrometry (LC/MS) is difficult because they are poorly ionized under electrospray ionization (ESI) conditions. The separation of short to very long chain saturated and unsaturated fatty acids is also very challenging when LC is employed instead of gas chromatography. The use of trimethylaminoethyl (TMAE) ester iodide derivatization has been shown previously to improve the sensitivities of saturated fatty acids in the ESI mode. A reversed-phase LC method using a diphenyl column was employed to separate 14 fatty acids as their TMAE derivatives. Stable isotope dilution LC/ESI/multiple reaction monitoring/MS methodology was then developed for the quantitative analysis of seven saturated and seven unsaturated forms of short (C14) to very long (C26) chain fatty acids as their TMAE ester iodide derivatives. This methodology has allowed the analysis of fatty acid composition from parental rat intestinal epithelial cell and rat intestinal epithelial cells transfected with cyclooxygenase-2, a model system of oxidative stress.