本文選題：高效液相色譜 + 氣相色譜串聯(lián)質(zhì)譜��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:建立高效液相色譜(HPLC)和氣相色譜串聯(lián)質(zhì)譜(GC-MS)的方法對(duì)肺癌細(xì)胞系A(chǔ)549細(xì)胞內(nèi)膽固醇濃度和肺組織內(nèi)27羥基膽固醇(27-OHC)水平有效進(jìn)行定性定量分析,為以細(xì)胞內(nèi)膽固醇含量作為指標(biāo)鑒定組織細(xì)胞泡沫化程度以及探討CYP27通路參與膽固醇代謝的后續(xù)研究奠定基礎(chǔ)。方法:A549細(xì)胞超聲裂解后,用KOH乙醇溶液和三氯乙酸分別除去細(xì)胞裂解液中的甘油三酯和蛋白,再以正己烷—異丙醇的二元提取劑將細(xì)胞內(nèi)的膽固醇進(jìn)行萃取。膽固醇含量采用HPLC法測(cè)定,色譜柱為ZORBAX SB-C18(4.6 mm×150 mm,5μm),流動(dòng)相為乙腈—異丙醇(67:33),流速為1 mL·min~(-1),檢測(cè)波長(zhǎng)為206 nm。定量稱取肺組織,施行高速勻漿法,獲得勻漿液。按勻漿液:氯仿(3:5)加入氯仿萃取。有機(jī)相用水沖洗直至中性,氯仿在減壓條件下移去。加入衍生化試劑0.1 ml三甲基氯硅烷/六甲基二硅氮烷/吡啶(1:2:3)60℃反應(yīng)60 min,氮?dú)獯蹈?氯仿復(fù)溶,進(jìn)GC/MS分析。色譜柱為HP-5MS石英毛細(xì)管柱,電離方式(EI)正離子掃描,單次進(jìn)樣1.0μl,載氣為氦氣。利用氣質(zhì)聯(lián)用方法分析肺組織內(nèi)27-OHC的水平。結(jié)果:利用本實(shí)驗(yàn)所建立的HPLC檢測(cè)方法,膽固醇標(biāo)準(zhǔn)品保留時(shí)間為8.2 min,5 ng·μL~(-1)—100 ng·μL~(-1)范圍內(nèi)呈良好的線性關(guān)系(R=0.9999);細(xì)胞內(nèi)膽固醇和半數(shù)量胞內(nèi)膽固醇出峰時(shí)間均與標(biāo)準(zhǔn)品溶液出峰時(shí)間一致,均在8.2 min處出峰。胞內(nèi)膽固醇定量檢測(cè),進(jìn)行方法學(xué)驗(yàn)證,其精密度、重復(fù)性、穩(wěn)定性均良好,RSD均低于3.0%,平均加樣回收率為94.89%,RSD為2.86%(n=6),檢測(cè)限為2.665ng·μL~(-1)(S/N=3)定量限為10.197 ng·μL~(-1)(S/N=10)。利用氣質(zhì)聯(lián)用方法檢測(cè),肺組織內(nèi)27羥基膽固醇碎片總離子流在55.39 min出峰,衍生化后的碎片質(zhì)荷比為m/z 456,m/z 546�？梢詫⑵涠ㄐ詼y(cè)出。對(duì)其定量檢測(cè),進(jìn)行方法學(xué)驗(yàn)證初步預(yù)測(cè),所測(cè)單次樣品的加樣回收率部分?jǐn)?shù)值在92%—105%范圍之外。其精密度、重復(fù)性、穩(wěn)定性等方法學(xué)驗(yàn)證指標(biāo)有待進(jìn)一步優(yōu)化。結(jié)論:本文所建立HPLC檢測(cè)的方法準(zhǔn)確、精密度高、重復(fù)性好、準(zhǔn)確度高、操作簡(jiǎn)便,回收率也在符合要求的范圍內(nèi),經(jīng)方法學(xué)驗(yàn)證,后續(xù)可用于A549細(xì)胞內(nèi)的膽固醇水平的批量檢測(cè);為進(jìn)階探討肺組織損傷與膽固醇代謝失調(diào)的病理進(jìn)展關(guān)系奠定基礎(chǔ)。氣相色譜串聯(lián)質(zhì)譜檢測(cè)肺組織內(nèi)27-OHC;該法目前可以有效定性檢測(cè)出組織內(nèi)27-OHC。但還需要進(jìn)一步進(jìn)行方法學(xué)論證。
[Abstract]:Objective: to establish a high performance liquid chromatography (HPLC) and gas chromatography-tandem mass spectrometry (GC-MS) method for the qualitative and quantitative analysis of cholesterol concentration in lung cancer cell line A549 and the level of 27 hydroxycholesterol (27-OHC) in lung tissue. The results provide a basis for identifying cellular foaming degree and further study on the involvement of CYP27 pathway in cholesterol metabolism. Methods after ultrasonic cleavage of cell line: A549 cells, cholesterol was extracted by Koh ethanol solution and trichloroacetic acid, respectively, after removing triglyceride and protein from the cell lytic solution, and then the cholesterol was extracted with a binary extractant of n-hexane-isopropanol. The cholesterol content was determined by HPLC. The chromatographic column was ZORBAX SB-C18 (4.6 mm 脳 150mm) mobile phase was acetonitrile-isopropanol (67:33), the flow rate was 1 mL min ~ (-1), the detection wavelength was 206 nm. The lung tissue was taken quantitatively and the homogenate was obtained by high speed homogenate method. According to homogenate: chloroform (3:5) added chloroform extraction. The organic phase is rinsed with water until neutral, and chloroform is removed under the condition of decompression. The derivative reagent 0.1 ml trimethylchlorosilane / hexamethyldisilyl azane / pyridine (1:2:3) was added for 60 min, then the nitrogen was blown dry, chloroform was redissolved and analyzed by GC / MS. The chromatographic column is HP-5MS quartz capillary column, the ionization mode (ei) is positive ion scanning, the single injection is 1.0 渭 l, the carrier gas is helium gas. The level of 27-OHC in lung tissue was analyzed by GC-MS. Results: the HPLC method was established. The retention time of cholesterol standard was 8.2 min ~ 5 ng 渭 L ~ (-1) -100 ng 渭 L ~ (-1) (R _ (0.9999), and the peak time of intracellular cholesterol and half amount of intracellular cholesterol was the same as that of standard solution, both of which were at 8.2 min. The accuracy, repeatability and stability were all lower than 3.0. The average recovery was 94.89% (n = 6) and the detection limit was 2.665ng 渭 L ~ (-1) (S / N ~ (3) = 10.197 ng / L ~ (-1) (S / N ~ (10). The total ion current of 27 hydroxyl cholesterol fragment in lung tissue was detected by GC-MS at 55.39 min, and the fragment mass charge ratio after derivation was m / z 456 m / z 546. It can be determined qualitatively. The quantitative detection and the preliminary prediction of methodology verification showed that the partial recovery of the single sample was beyond 92% -105%. Its precision, repeatability, stability and other methodological verification indicators need to be further optimized. Conclusion: the HPLC method established in this paper is accurate, reproducible, accurate, easy to operate, and the recovery rate is within the range of requirements. The follow-up can be used for batch detection of cholesterol level in A549 cells, which lays a foundation for the advanced study of the relationship between lung tissue injury and cholesterol metabolism disorder. Gas chromatography-tandem mass spectrometry (GC-MS) was used to detect 27-OHC27 in lung tissue. However, further methodological arguments are needed.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

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