Abstract：
An UPLC-APCI-MS/MS method was developed for the simultaneous determination of cholesterol, 7-dehydrocholesterol (7DHC) and eight oxysterols including 27-hydroxycholesterol (27OHC), 7α-hydroxycholesterol (7αOHC), 7β-hydroxycholesterol (7βOHC), 24S-hydroxycholesterol (24SOHC), 25-hydroxycholesterol (25OHC), 7α,24S-dihydroxycholesterol (7α,24SdiOHC), 7α,25-dihydroxycholesterol (7α,25diOHC), and 7α,27-dihydroxycholesterol (7α,27diOHC). It has been used for quantitative analysis of cholesterol, 7DHC and eight oxysterols in hepatocellular carcinoma (HCC) cells, plasma and tumor tissue samples. And the above compounds were extracted from the biological matrix (plasma and tissue) using liquid-liquid extraction with hexane/isopropanol after saponification to cleave the steroids from their esterified forms without further derivatization. Then cholesterol, 7DHC and oxysterols were separated on a reversed phase column (Agilent Zorbax Eclipse plus, C18) within 8 min using a gradient elution with 0.1 % formic acid in H2O and methanol and detected by an APCI triple quadrupole mass spectrometer. The lower limit of quantification (LLOQ) of the cholesterol, 7DHC and oxysterols ranged from 3.9 ng/mL to 31.25 ng/mL, and the recoveries ranged from 83.0 % to 113.9 %. Cholesterol, 7DHC and several oxysterols including 27OHC, 7αOHC and 7βOHC were successfully quantified in HCC cells, plasma, tissues and urine of HCC mice. Results showed that 27OHC was at high levels in three kind of HCC cells and tumor tissues as well as plasma samples from both HepG2 and Huh7 bearing mice model，and the high levels of 27OHC in tumors were associated with HCC development. Moreover, the levels of cholesterol in HCC cells and tumor issues varied in different HCC cells and mice model. Oxysterols profiling in biological samples might provide complementary information in cancer diagnosis.
摘要：
建立了UPLC-APCI-MS/MS同时测定胆固醇的方法,7-脱氢胆固醇（7DHC）和八种氧固醇，包括27-羟基胆固醇，7α-羟基胆固醇（7αOHC），7β-羟基胆固醇（7βOHC），24S-羟基胆固醇（24SOHC），25-羟基胆固醇（25OHC），7α,24S-二羟基胆固醇（7α，24SdiOHC），7α,25-二羟基胆固醇（7α，25diOHC），和7α，27-二羟基胆固醇（7α，27diOHC）。它已被用于胆固醇的定量分析，肝细胞癌（HCC）细胞中的7DHC和八种氧固醇，血浆和肿瘤组织样本。皂化后，使用己烷/异丙醇液-液萃取从生物基质（血浆和组织）中提取上述化合物，以将类固醇从其酯化形式裂解而无需进一步衍生化。然后是胆固醇，7DHC和氧固醇在反相柱(AgilentZorbaxEclipseplus，C8）在8min内使用在H2O和甲醇中的0.1％甲酸进行梯度洗脱，并通过APCI三重四极杆质谱仪进行检测。胆固醇的定量下限（LLOQ），7DHC和氧固醇的范围为3.9ng/ml至31.25ng/ml，回收率在83.0%至113.9%之间。胆固醇,7DHC和几种氧固醇，包括27OHC，7αOHC和7βOHC在HCC细胞中成功定量，等离子体,肝癌小鼠的组织和尿液。结果表明，在HepG2和Huh7小鼠模型的三种HCC细胞和肿瘤组织以及血浆样品中，27OHC的水平均较高，并且肿瘤中27OHC的高水平与HCC的发展有关。此外，在不同的HCC细胞和小鼠模型中，HCC细胞中的胆固醇水平和肿瘤问题有所不同。生物样品中的氧化固醇谱可能为癌症诊断提供补充信息。
