LC-MS_

∗基金项目:国家自然科学基金资助项目(编号:82073676);北京市属医学科研院所公益发展改革试点项目(编号:京医研2021-10)
作者单位:100069北京市首都医科大学附属北京佑安医院/北京肝病研究所/北京市肝炎与肝癌精准医疗及转化工程技术研究中心
第一作者:蔡玉莹,女,26岁,理学硕士,技师㊂主要从事代谢小分子质谱研究㊂E-mail:caiyuying0113@
通讯作者:陈德喜,E-mail:dexichen@ ㊃实验性肝炎㊃
LC-MS/MS定量分析小鼠肝组织15种胆汁酸成分研究∗
蔡玉莹,殷继明,宁琪琪,高玉雪,杨鹏翔,陈德喜
㊀㊀ʌ摘要ɔ㊀目的㊀建立快速高效的液相色谱-串联质谱法(LC-MS/MS)同时测定小鼠肝组织15种胆汁酸(BAs)浓度㊂方法㊀采用活性炭制备无BAs的空白肝组织,作为制备标准样品和质量控制样品的生物基质㊂匀浆小鼠肝组织,加入碱性乙腈溶液(5%NH4OH))沉淀蛋白㊂以2H4-DCA,GUDCA-d5和LCA-d4为内标物,用Agilent Poroshell120EC C18色谱柱(100mmˑ4.6mm,2.7μm)分离,以醋酸铵水溶液和甲醇-乙腈溶液为流动相梯度洗脱,柱温30ħ,流速0.3mL/min,进样量为2μL,采用电喷雾离子源(ESI)负离子模式,多反应监测(MRM)㊂结果㊀15种BAs线性关系良好,R2均大于0.993,定量检测限均小于2ng/mL,基质效应为90.76%~109.25%;日内㊁日间准确度和精密度均小于15%,4ħ24h㊁反
复冻融㊁冷冻保存1个月稳定性良好,满足生物样品的分析要求;小鼠肝组织检测结果显示游离型BAs和结合型BAs(G-BAs㊁T-BAs)都以母体CA为主,TCA含量最高;游离BAs浓度为(723.89ʃ50.65)ng/mL,显著高于G-BAsʌ(56.90ʃ
11.28)ng/mL,P<0.001ɔ,T-BAs浓度为(40322.90ʃ14034.80)ng/mL,显著高于游离BAs(P<0.001)或G-BAs(P<
0.001)㊂结论㊀建立的LC-MS/MS方法灵敏度高,准确可靠,适用于检测小鼠肝组织BAs浓度㊂
㊀㊀ʌ关键词ɔ㊀肝组织;胆汁酸;液相色谱-串联质谱;小鼠
㊀㊀DOI:10.3969/j.issn.1672-5069.2023.03.005
㊀㊀Quantitative profiling of15bile acids in mouse liver tissues by using liquid chromatography-tandem mass spectrometry㊀Cai Yuying,Yin Jiming,Ning Qiqi,et al.Beijing Institute of Hepatology,Beijing Precision Medicine and Transformation Engineering,Technology Research Center of Hepatitis and Liver Cancer,You an Hospital Affiliated to Capital Medical University, Beijing100069,China
㊀㊀ʌAbstractɔ㊀Objective㊀The purpose of this study was to establish a rapid and efficient liquid chromatography tandem mass spectrometry(LC-MS/MS)for simultaneous determination of15bile acids in mouse liver tissues.Methods㊀The activated charcoal was utilized to prepare bile acid-free liver,which served as the biological matrix for the preparation of standard and quality control samples.The mouse liver tissue was homogenized,and a basic acetonitrile solution,including5%NH4OH was added to precipitate proteins.The proteins were separated on an Agilent Poroshell120EC C18column(100mmˑ4.6mm,2.7μm)by using2H4-DCA,GUDCA-d5,and LCA-d4as internal standards.The mobile phase is ammonium acetate aqueous solution and methanol acetonitrile mixed solution for gradient elution,the column temperature was30ħ,the flow rate was0.3mL/min,and the injection volume was2μL.The electrospray ion source(ESI)was operated in negative ion mode,and in multiple reaction monitoring(MRM).Results㊀The linearity of the15bile acids was good with R2greater than0.993,the limits of determination were less than2ng/mL,and the matrix effects were90.76%-109.25%;the intra-day and inter-day accuracy and precision were less than15%,and the stability was good under4ħfor24h,repeated freeze-thaw,and freeze-storage for one month,meeting the analytical requirements of biological samples;the detection of mouse liver tissues showed that both unconjugated BAs and conjugated BAs(G-BAs,T-BAs)were dominated by maternal CA,with the highest content of TCA;the concentration of unconjugated BAs was(723.89ʃ50.65)ng/mL,significantly higher than that of G-BAs[(56.90ʃ11.28)ng/mL,P<0.001];
the concentration of T-BAs was(40322.90ʃ14034.80)ng/mL,
significantly higher than unconjugated BAs(P<0.001),and
also significantly higher than G-BAs(P<0.001).Conclusion
㊀The LC-MS/MS method we established is sensitive,accurate,
reliable,and suitable for the determination of bile acids
concentrations in mouse liver tissues,which might help for
further studies.
㊀㊀ʌKey wordsɔ㊀Liver tissues;Bile acids;Liquid
chromatography-tandem mass spectrometry;Mouse
㊀㊀胆汁酸(bile acids,BAs)是一类二十四碳胆烷酸羟基衍生物的总称,在胆固醇稳态㊁脂质吸收和肠道信号传导过程中发挥重要作用[1,2]㊂肝脏中合成的主要BAs是胆酸(cholic acid,CA)和鹅去氧胆酸(chenodeoxycholic acid,CDCA)[3]㊂在肠肝循环过程中,一部分初级BAs被微生物菌群转化为次级BAs,如脱氧胆酸(deoxycholic acid,DCA)㊁石胆酸(litho-cholic acid,LCA)和熊去氧胆酸(ursodeoxycholic acid,UDCA)[4-7]㊂目前,检测BAs的方法有酶法[8-11]㊁薄层色谱法(thin-layerchromatography, TLC)[12-14]㊁气相色谱-质谱联用法(gas chromatography-mass spectrometer,GC-MS)[15]㊁高效液相色谱法(high performance liquid chromatography, HPLC)[16,17]和液相色谱-质谱联用法(liquid chroma-tography tandem mass spectrometry,LC-MS/MS)等[18]㊂本研究建立了一种快速灵敏的LC-MS/MS 法,无需衍生化,分析速度快,高通量,可以同时测定小鼠肝组织15种BAs,为科研提供了技术支持㊂
1㊀材料与方法
1.1仪器㊁试剂与动物㊀Agilent6495三重四极杆质
谱仪系统㊁Agilent1290型超高效液相色谱仪系统(美国Agilent公司);BSA224S型微量电子天平(德
国Sartorius公司);MS3Digital型涡旋混匀仪(德国IKA公司);Centrifuge5424R型高速离心机(德国Eppendorf公司);高速低温组织研磨仪(中国武汉塞维尔生物科技有限公司)㊂Milli-Q Advantage纯水仪(美国Millipore公司)制备超纯水(18.2MΩ㊃cm);CA㊁DCA㊁CDCA㊁UDCA㊁LCA㊁甘氨去氧胆酸(glycodeoxycholic Acid,GDCA)㊁甘氨鹅去氧胆酸(glycochenodeoxycholic Acid,GCDCA)㊁甘氨熊去氧胆酸(glycoursodeoxycholic Acid,GUDCA)㊁牛磺胆酸(taurocholate Acid,TCA)㊁牛磺去氧胆酸(taurodeoxy-cholic Acid,TDCA)㊁牛磺鹅去氧胆酸(taurochenode-oxycholic Acid,TCDCA)㊁牛磺熊去氧胆酸(taurour-sodeoxycholic Acid,TUDCA)㊁牛磺石胆酸(taurolitho-cholic Acid,TLCA)标准品及三个稳定同位素标记的内标物质(internal standard,IS)石胆酸-d4 (lithocholic acid-d4,LCA-d4)㊁甘氨熊脱氧胆酸-d5 (glycoursodeoxycholic Acid-d5,GUDCA-d5)㊁2H4-脱氧胆酸(2H4-deoxycholic acid,2H4-DCA)(中国上海安谱实验科技有限公司);甘氨胆酸(glycocholic Acid,GCA)㊁甘氨石胆酸(glycolithocholic acid, GLCA)购自加拿大TRC公司㊂甲醇㊁乙腈(色谱纯, Thermo Fisher公司),醋酸盐(美国Sigma-Aldrich公司),活性炭(美国Sigma-Aldrich公司)㊂雄性SPF 级C57BL/6小鼠,5~9周龄,体质量为24~32g,由北京维通达生物技术有限公司提供㊂严格遵守首都医科大学动物伦理和福利相关规定(伦理批件号: AEEI-2021-257)进行动物实验㊂
1.2标准溶液的配制㊀称取标准品,用甲醇溶解配制成质量浓度均为1mg/mL单标储备液㊂以不同比例准确移取各标准品溶液,制成混合标准溶液㊂用甲醇稀释配制成11个浓度梯度标准溶液以及低㊁中㊁高三个浓度质控溶液,其中CA㊁DCA㊁CDCA㊁UDCA㊁TCA㊁TDCA㊁TCDCA㊁TUDCA浓度为3.9ng/mL~ 4μg/mL,质控浓度分别为40ng/mL㊁400ng/mL㊁3μg/mL;GCA㊁GDCA㊁GCDCA㊁GUDCA浓度为1.95 ng/mL~2μg/mL,质控浓度分别为20ng/mL㊁200 ng/mL㊁1.5μg/mL;LCA㊁GLCA㊁TLCA浓度为0.98 ng/mL~1μg/mL,质控浓度分别为10ng/mL㊁100 ng/mL和1μg/mL㊂IS浓度为1μg/mL㊂
1.3色谱条件㊀色谱柱:Agilent Poroshell120EC C18色谱柱(100mmˑ4.6mm,
2.7μm),流动A相为5 mmol/L醋酸铵水溶液,B相为乙腈/甲醇比例为3比2的混合溶液;梯度洗脱程序:起始40%B,0~2 min40%B;2~8min60%B;8~10min95%B;10~ 13min95%B维持;13~15min40%B㊂流速为0.3 mL/min,柱温30ħ,自动进样器温度4ħ,进样器体积为2μL㊂
1.4质谱条件㊀采用Agilent6495三重四极杆质谱系统和电喷雾电离(electron spray ionization,ESI)负离子模式,多重反应监测(multiple reaction monitoring, MRM),Nebuilzer20psi,Gas Flow14L/min,Sheath Gas Temp250ħ,sheath gas flow11L/min,capillary 3000V,nozzle voltage1500V㊂
1.5肝组织BAs检测㊀精密称取肝组织90mg,加去离子水500μL,加研磨珠匀浆㊂将肝组织以60HZ 匀浆10min,取50μL匀浆液加入-20ħ预冷的碱性乙腈(5%NH4OH)250μL,加入IS(1μg/mL2H4-DCA,1μg/mLGUDCA-d5,1μg/mL LCA-d4)10μL,涡旋30min,15000r/m离心10min㊂吸取上清液100μL,加入内插管中,注入LC-MS/MS系统进行分析㊂
1.6空白基质处理㊀称取3只普通小鼠肝组织90mg,加去离子水500μL匀浆㊂加入活性炭(100mg/mL)500μL孵育30min,去除基质中的内源性BAs㊂将混合物在16000g下离心10min,取出上清液600μL,过滤,在16000g离心1min㊂去除基质滤液,以构建肝脏生物基质的标准曲线㊂
2㊀结果
2.1质谱条件的优化情况㊀采用1μg/mL的单标准品溶液,流速为0.30mL/min,对15种BAs质谱参数进行优化㊂应用ESI源,在MRM模式下对标准品及其IS的母离子㊁子离子和碰撞能量进行逐一优化㊂
结果表明,在ESI 负离子模式下,15种BAs 和3种IS 都可以获得强度较高的[M -H]-准分子离子峰,在二级质谱高碰撞能量下获得丰度较高的子离子㊂对于游离BAs,使用母离子定量,对甘氨结合型胆汁酸(glycine -conjugated bile acids,G -BAs)以73.9m /z (NHCH 2COOH -)为子离子,对牛磺结合型胆汁酸(taurine -conjugated bile acids,T -BAs)以79.8m /z (SO 3-)为子离子㊂继续优化碰撞能量,使目标化合物具有最高的灵敏度㊂
2.2色谱条件的优化情况㊀本实验结果发现,流动A 相为5mmol /L 醋酸铵水溶液,B 相为乙腈甲醇(3ʒ2)的混合溶液,采用梯度洗脱时,能够很好地分离各类BAs,且各物质峰形良好,具有较高的灵敏度(图1)㊂醋酸铵的加入极大地提高了离子响应,将分析时间缩短到15min,能够快速高效地分析15种BAs,确保母离子和子离子都相同的BAs 能实现基线分离,达到很好的定量分析效果
㊂
图1㊀15种BAs 的色谱图
横坐标为采集时间,纵坐标为counts (%),15种BAs 的峰型良好
游离BAs (以红色显示),其中1:CA;2:DCA;3:CDCA;4:UDCA;5:LCA
G -BAs(以蓝色显示),其中6:GCA;7:GDCA;8:GCDCA;9:GUDCA;10:GLCA
T -BAs (以黑色显示),其中11:TCA;12:TDCA;13:TCDCA;14:UDCA;15:TLCA
2.3样本前处理的优化㊀BAs 属于内源性物质,无法直接从小鼠肝组织提取以作为空白基质㊂采用模拟空白基质与实际背景相差较大,造成检测误差㊂因此,在本研究,采用活性炭吸附法去除小鼠肝组织内源性BAs 的干扰,以获得较为准确的空白基质㊂预实验结果表明吸附后的肝组织匀浆溶液均无各类BAs 检出㊂在已发表的研究,对肝组织前处理一般使用SPE 固相萃取等方法,但这种方法耗时长㊁成本高,不适用于大批量样本检测㊂本实验采用蛋白沉淀法,样本经碱性乙腈处理后离心沉淀,简化了前处理过程,提高了检测效率㊂
2.4标准曲线和检出限的确定㊀以活性炭处理后的空白基质为背景,加入15个BAs 和3个IS,在适当范围构建11个校准点的标准曲线㊂以分析物与IS
的峰面积比为纵坐标,各标准品浓度为横坐标作标准曲线,通过调整最佳加权因子1/X 或1/X 2(X 是每种分析物的浓度)分析校准㊂结果表明,15种BAs 线性良好,R 2均大于0.993㊂以信噪比S /N =3ʒ1时对应的浓度确定本方法的检出限(limit of detection,LOD),LOD 均小于2ng /mL㊂
2.5精密度㊁准确度和基质效应情况㊀取空白肝组织匀浆液和各BAs 标准品溶液,制备15种BAs 低㊁中㊁高浓度质控样本,每一浓度平行操作6份,1天内进样6次,以获得日内检测结果,3日重复进样,获得日间检测结果,并代入线性回归方程,计算浓度㊂准确度计算方法为:准确度(RE)=测算值-真实值/真实值ˑ100%;精密度计算方法为:相对标准偏差(RSD)=标准偏差(SD)/计算结果的算术平均值(X)ˑ100%㊂在本方法中,15种BAs 在低㊁中㊁高QC 样本浓度下日内RE 为-1
3.18%~10.95%,日内RSD 为0.92%~11.74%;日间RE 为-11.27%~8.58%,日间RSD 为2.07%~11.33%㊂通过比较在甲醇标准溶液和空白基质溶液中,以15种BAs 低㊁中㊁高QC 样本的比值来确定基质效应㊂在本方法中,基质效应值为90.76%~109.25%㊂测定结果表明,小鼠肝组织BAs 分析的准确度㊁精密度和基质效应良好㊂2.6稳定性㊀取空白肝组织匀浆液和各BAs 标准品溶液,制备15种BAs 低㊁中㊁高浓度各6份标本,分别置于自动进样器4ħ24h,反复冻融3次,-80ħ冷冻保存1个月,测定结果显示在自动进样器4ħ存放24h 时,RE 为-12.67%~7.74%,RSD 为0.71%~13.28%;反复冻融3次,RE 为-13.98%~10.75%,RSD 为0.67%~15.53%;-80ħ冷冻保存1个月,RE 为-13.89%~12.89%,RSD 为0.62%~15.32%㊂在以上三种测定条件下,样品较稳定,未见明显降解,表明在这些条件下样本均能保持稳定㊂2.7肝组织BAs 测定㊀应用上述所建立的方法测定6例小鼠肝组织15种BAs 含量,游离BAs 主要以初级BAs 的CA 和CDCA 为主,次级BAs 的DCA 和LCA 含量较低(图2A);G -BAs 以GCA 为主,甘氨酸结合型浓度较低,在线性范围内检测不到GCDCA 和GUDCA(图2B);小鼠肝组织T -BAs 主要以TCA 和TDCA 为主(图2C );游离BAs 浓度[(723.89ʃ50.65)ng /mL],显著高于G -BAs[(56.90ʃ11.28)ng /mL,P <0.001];T -BAs 浓度[(40322.90ʃ1403
4.80)ng /mL]显著高于游离BAs[(723.89ʃ50.65)ng /mL,P <0.001],也显著高于G -BAs [(56.90ʃ11.28)ng /mL,P <0.001,图2D],提示小鼠肝组织BAs 代谢水平的差异性和复杂性,值得深入研究㊂3　讨论
本研究基于靶向LC -MS /MS 技术建立了检测小
图2㊀LC-MS/MS检测6例小鼠肝组织BAs浓度
A:各游离BAs浓度;B:各G-BAs浓度;C:各T-BAs 浓度;D:游离BAs㊁G-BAs和T-BAs浓度
游离BAs与G-BAs相比,∗∗∗P<0.001;游离BAs 与T-BAs相比,∗∗∗P<0.001;G-BAs与T-BAs相比,∗∗∗P<0.001
鼠肝组织15种BAs含量的检测方法,并进行了方法学验证㊂使用Agilent Poroshell120EC C18色谱柱, ESI离子源,流动相中醋酸铵的加入,在负离子MRM 模式下提高了离子化效率,改善了峰形㊂而过高的醋酸铵则会抑制BAs的离子化,前期实验表明5 mmol/L醋酸铵离子化效果最佳㊂
BAs属于小鼠肝脏的内源性物质,无法直接获取空白基质㊂本实验中通过活性炭吸附,有效去除内源性BAs,最大程度降低对检测结果的影响,使准确性显著提高㊂采用乙腈蛋白沉淀的前处理方法,仅需50μL样本,操作简单快速,背景干净,基质效应良好㊂实验结果表明,15种BAs在其线性浓度范围内线性相关系数R2均大于0.993,LOD均小于2 ng/mL,基质效应范围为90.76%~109.25%㊂准确度㊁精密度和稳定性均满足生物样本分析要求㊂检测小鼠肝组织BAs,结果TCA和TDCA浓度较高㊂准确定量BAs亚型浓度对进一步研究肝脏疾病具有重要意义㊂本研究采用LC-MS/MS检测了小鼠肝组织15种BAs含量,结果显示游离BAs中的初级BAs CA和CDCA浓度分别是对应次级BAs DCA和LCA的22倍和31倍,且初级BAs中CA是CDCA的1.25倍,符合BAs的合成过程㊂游离型BAs和结合型BAs(G-BAs㊁T-BAs)都以母体CA为主,其中TCA浓度最高,是CA的115倍或GCA的683倍㊂6例样本的BAs含量存在一定的差异,尤其是T-BAs,除小鼠个体差异外,还可能是采集时胆汁的污染造成的㊂T-BAs是小鼠的主要BAs,符合已有研究结果,与人类或大鼠不同㊂在小鼠,负责BAs与氨基酸结合的酶对牛磺酸具有更强的亲和力㊂
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(收稿:2022-11-10)
(本文编辑:陈从新)。