CN111812264B - 一种去氧胆酸类化合物的生物样品分析方法 - Google Patents
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Abstract
本发明提供了一种去氧胆酸类化合物的生物样品分析方法,是通过高效、快速、灵敏的液相‑质谱联用系统(LC‑MS)方法对去氧胆酸类化合物进行分析。此方法采取了简单的有机溶剂蛋白沉淀法对生物样本进行前处理,基于待测物与内标的峰面积比,通过归一化法得到标准曲线,从而定量得到生物样品中去氧胆酸类化合物的浓度。本法为去氧胆酸类化合物在动物体内的药代动力学中去氧胆酸类化合物的检测提供了简便的分析方法。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种去氧胆酸类化合物的生物样品分析方法。
背景技术
法尼醇X受体(FXR)是一种核受体,是一种胆酸的传感器,可调控胆酸的合成和胆汁在肝内的流动,同时对于胆汁内环境稳定、脂质代谢、糖代谢以及炎症/免疫应答都有作用。
研究表明,鹅去氧胆酸的6位取代衍生物,是半合成的鹅去氧胆酸(CDCA)的衍生物,可激活法尼醇X受体(FXR)。6-α-乙基鹅去氧胆酸(6-ECDCA,OCA)对于FXR的激活作用是CDCA的100倍,临床研究表明,OCA可以用于治疗原发性胆汁性肝硬化(PBC)、门静脉高压(Portal hypertension)、非酒精性脂肪肝炎(NASH)、胆汁酸性腹泻(Bile aciddiarrhea)、酒精性肝炎、原发性硬化性胆管炎(PSC)等与胆汁分泌有关的疾病(DrugDiscovery Today.Volume 17,Numbers 17/18,2012)。然而,去氧胆酸类化合物(如CDCA、UDCA或OCA),在pH值为1~8的环境下不溶于含水介质,具有极强的苦味,并且苦味持续数小时。CDCA和OCA的pKa值均为5,显弱酸性,在水中的溶解度很低,其中,CDCA在水中的溶解度约为32μM,而OCA为9μM(J.Pharmacol.Exp.Ther.July2014.350:56-68)。UDCA水中溶解度为53μM,并且只有当pH值达到8.47才会出现完全溶解,而当pH值小于8.4时,部分熊去氧胆酸不但不能被吸收,反而被肠道细菌菌落转化成石胆酸(US5380533)。而如中国专利CN108218946A所公开的去氧胆酸类化合物,能屏蔽奥贝胆酸的苦味或对胃肠道的刺激,水溶性好,生物利用度高,可在肠道中释放出奥贝胆酸,从而具备相同的药理活性、更佳的药代动力学性质。在制备预防和/或治疗与胆汁分泌有关的疾病如胆固醇性胆结石症、原发性胆汁性肝硬化、门静脉高压、非酒精性脂肪肝炎、胆汁酸性腹泻、酒精性肝炎、原发性硬化性胆管炎或动脉粥样硬化的药物中有着潜在的应用前景。然而目前去氧胆酸类化合物在生物样品中浓度的检测方法存在操作步骤复杂、分析周期长以及生物基质取材单一等问题。
发明内容
本发明针去氧胆酸类化合物在生物样品中浓度的检测方法存在操作复杂、分析周期长以及生物基质取材单一的问题,提供了一种去氧胆酸类化合物的生物样品分析方法。本检测方法操作简单,分析快速,而且能应用于多种不同的生物基质。
为了解决上述技术问题,本发明采取的技术方案之一为:一种去氧胆酸类化合物的生物样品分析方法,该分析方法包括以下步骤:
(1)标准曲线溶液配制
称取10.73mg去氧胆酸类化合物对照品,置于100ml容量瓶中,加乙腈溶解并稀释至刻度,摇匀后配制成0.1073mg/ml的标准溶液,再次加入乙腈稀释,配制成浓度分别为1-1000ng/mL的标准曲线溶液;
(2)质控溶液配制
称取10.94mg去氧胆酸类化合物对照品,置于100ml容量瓶中,加乙腈溶解并稀释至刻度,摇匀后配制成0.1094mg/ml的质控溶液,再次加入乙腈稀释,配制成浓度分别为2,100,800ng/mL的质控溶液;
(3)内标溶液配制
称取11.29mg非诺贝特酸-d6对照品,置于100ml容量瓶中,加乙腈溶解并稀释至刻度,摇匀后配制成0.1129mg/ml的内标溶液,再次加入乙腈稀释,配制成浓度为100ng/mL的内标溶液;
(4)生物样品前处理
取生物样品,加入重量:体积比为1g:6mL的生理盐水匀浆,取100μL匀浆后的生物样品至1.5mLEP管中,加入100μL浓度为100ng/mL的非诺贝特酸-d6内标溶液,混匀,加入300-500μL乙腈沉淀,涡旋2-3分钟混匀,4℃,13000-15000rpm离心15-20分钟,取200μL上清液进样;
(5)LC-MS/MS方法检测
通过LC-MS/MS方法检测,基于待测物与内标的峰面积比,通过归一化法得到标准曲线,从而定量得到生物样品中去氧胆酸类化合物的浓度。
优选地,所述LC-MS/MS方法的色谱及质谱条件如下:
色谱条件:液相型号为岛津LC-20A;色谱柱为Agela Venusil XBP C-18;流动相A相为5mM乙酸铵水溶液,流动相B相为0.1%甲酸乙腈溶液,A:B=15:85;
质谱条件:API 4000(ESI源)质谱,负离子,离子喷射电压为-4500V,温度500℃,雾化气压力为50psi,解簇电压压力为50psi,气帘气体压力为20psi,扫描方式为多重反应监测(MRM)。
优选地,所述生物样品选自受试者的血液或血清样品。
优选地,所述去氧胆酸类化合物具有如下通式结构:
其中,
R1为“-NR2R3取代的C1~C4烷基”、C15~C21烷基或C15~C21烯基;
R2为氢或C1~C10烷基,R3为氢或C1~C10烷基;或者,R2、R3及与它们相连接的N原子共同形成4~8元杂环基。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:本检测方法是通过高效、快速、灵敏的液相-质谱联用系统(LC-MS)方法对生物样品中的去氧胆酸类化合物进行分析。此方法采取了简单的有机溶剂蛋白沉淀法对生物样本进行前处理,基于待测物与内标的峰面积比,通过归一化法得到标准曲线,从而定量得到生物样本中去氧胆酸类化合物的浓度。本法为去氧胆酸类化合物在动物体内的药代动力学中去氧胆酸类化合物的检测提供了简便的分析方法。
附图说明
图1为去氧胆酸类化合物及内标的色谱图,其中图1(A)为去氧胆酸类化合物色谱图,图1(B)为内标色谱图。
图2为基于待测物与内标的峰面积比,通过归一化法得到的标准曲线。
具体实施方式
以下的实施例用于为本领域中的普通技术人员提供有关如何实施和使用本发明的完整披露和描述,并且这些例子并非意在对发明人所认为的发明范围进行限制,亦非意指下文的实验是被实施的全部实验而且是仅可实施的实验。实验例中未注明具体条件的实验方法,通常按照常规条件或按照厂商所建议的条件。
本发明所使用的仪器和材料如下:
仪器与试剂:高效液相色谱仪:岛津LC-20A HPLC(日本岛津公司),DAD检测器(美国安捷伦公司);色谱柱:Agela Venusil XBP C-18(150×4.6mm,5μm,天津博纳艾杰尔科技有限公司);非诺贝特酸-d6标准品(TLC Pharmaceutical Standards);乙腈、乙酸铵、及甲酸乙腈溶液均购于国药集团。
实施例1检测生物样品中去氧胆酸类化合物浓度
(1)标准曲线溶液配制
称取10.73mg去氧胆酸类化合物对照品,置于100ml容量瓶中,加乙腈溶解并稀释至刻度,摇匀后配制成0.1073mg/ml的标准溶液,再次加入乙腈稀释,配制成浓度分别为1-1000ng/mL的标准曲线溶液;
(2)质控溶液配制
称取10.94mg去氧胆酸类化合物对照品,置于100ml容量瓶中,加乙腈溶解并稀释至刻度,摇匀后配制成0.1094mg/ml的质控溶液,再次加入乙腈稀释,配制成浓度分别为2,100,800ng/mL的质控溶液;
(3)内标溶液配制
称取11.29mg非诺贝特酸-d6对照品,置于100ml容量瓶中,加乙腈溶解并稀释至刻度,摇匀后配制成0.1129mg/ml的内标溶液,再次加入乙腈稀释,配制成浓度为100ng/mL的内标溶液;
(4)生物样品前处理
取生物样品,加入重量:体积比为1g:6mL的生理盐水匀浆,取100μL匀浆后的生物样品至1.5mL EP管中,加入100μL浓度为100ng/mL的非诺贝特酸-d6内标溶液,混匀,加入300-500μL乙腈沉淀,涡旋2-3分钟混匀,4℃,13000-15000rpm离心15-20分钟,取200μL上清液进样;
(5)LC-MS/MS方法检测
色谱条件:液相型号为岛津LC-20A;色谱柱为Agela Venusil XBP C-18;流动相A相为5mM乙酸铵水溶液,流动相B相为0.1%甲酸乙腈溶液,A:B=15:85;
质谱条件:API 4000(ESI源)质谱,负离子,离子喷射电压为-4500V,温度500℃,雾化气压力为50psi,解簇电压压力为50psi,气帘气体压力为20psi,扫描方式为多重反应监测(MRM)。
源参数如表1所示:
表1
化合物参数如表2所示:
表2
(6)色谱图获得
经过上述步骤进样分析得到去氧胆酸类化合物及内标的色谱图,如图1所示。其中图1(A)为去氧胆酸类化合物色谱图,图1(B)为内标色谱图。
(7)标准曲线
基于待测物与内标的峰面积比,通过归一化法得到标准曲线,如图2所示。
(8)定量结果
通过标准曲线定量得到生物样品中去氧胆酸类化合物的浓度如表3所示:
表3
时间点(h) | 浓度(ng/mL) |
0.25 | 833 |
0.5 | 752 |
1 | 660 |
3 | 254 |
5 | 88.1 |
7 | 23.8 |
12 | 3.24 |
24 | BLOQ |
虽然在实施例中已经通过一般性说明、具体实施方式及试验对本发明作出了详尽的描述,但在不偏离本发明核心的基础上,仍可以作出的修改或改进,均属于本发明要求保护的范围。
Claims (2)
1.一种去氧胆酸类化合物的生物样品分析方法,其特征在于,该分析方法包括以下步骤:
(1)标准曲线溶液配制
称取10.73 mg去氧胆酸类化合物对照品,置于100 ml容量瓶中,加乙腈溶解并稀释至刻度,摇匀后配制成0.1073 mg/ml的标准溶液,再次加入乙腈稀释,配制成浓度分别为3,5,10,30,100,300,1000、2000 ng/mL的标准曲线溶液;
(2)质控溶液配制
称取10.94 mg去氧胆酸类化合物对照品,置于100 ml容量瓶中,加乙腈溶解并稀释至刻度,摇匀后配制成0.1094 mg/ml的质控溶液,再次加入乙腈稀释,配制成浓度分别为5,10,1500 ng/mL的质控溶液;
(3)内标溶液配制
称取11.29 mg地西泮对照品,置于100 ml容量瓶中,加乙腈溶解并稀释至刻度,摇匀后配制成0.1129 mg/ml的内标溶液,再次加入乙腈稀释,配制成浓度为100 ng/mL的内标溶液;
(4)生物样品前处理
取生物样品,加入重量:体积比为1g : 6 mL的生理盐水匀浆,取100 μL匀浆后的生物样品至1.5 mL EP管中,加入100μL浓度为100 ng/mL的地西泮内标溶液,混匀,加入300-500μL乙腈沉淀,涡旋2-3分钟混匀,4℃,13000-15000 rpm离心15-20分钟,取200 μL上清液进样;
(5)LC-MS/MS方法检测
通过LC-MS/MS方法检测,基于待测物与内标的峰面积比,通过归一化法得到标准曲线,从而定量得到生物样品中去氧胆酸类化合物的浓度,
LC-MS/MS方法的色谱及质谱条件如下:
色谱条件:液相型号为岛津 LC-20A;色谱柱为Agela Venusil XBP C-18;流动相A相为5mM 乙酸铵水溶液,流动相B相为0.1%甲酸乙腈溶液,A:B=15:85;
质谱条件:API 4000质谱ESI源,负离子,离子喷射电压为5500 V,温度570℃,雾化气压力为52 psi,解簇电压压力为58 psi,气帘气体压力为28 psi,扫描方式为多重反应监测;
所述去氧胆酸类化合物具有如下通式结构:
其中,
R1为“-NR2R3取代的C1~C4烷基”、C15~C21烷基或C15~C21烯基;
R2为氢或C1~C10烷基,R3为氢或C1~C10烷基;或者,R2、R3及与它们相连接的N原子共同形成4~8元杂环基。
2.如权利要求1所述去氧胆酸类化合物的生物样品分析方法,其特征在于,所述生物样品选自受试者的血液或血清样品。
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