WO2022110569A1 - 一种大鼠血浆中芦荟苦素的lc-ms/ms测定方法 - Google Patents

一种大鼠血浆中芦荟苦素的lc-ms/ms测定方法 Download PDF

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WO2022110569A1
WO2022110569A1 PCT/CN2021/080459 CN2021080459W WO2022110569A1 WO 2022110569 A1 WO2022110569 A1 WO 2022110569A1 CN 2021080459 W CN2021080459 W CN 2021080459W WO 2022110569 A1 WO2022110569 A1 WO 2022110569A1
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aloin
mobile phase
rat plasma
aloe
internal standard
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PCT/CN2021/080459
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French (fr)
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谭银丰
李海龙
李友宾
王雪松
崔雪
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海南医学院
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Priority to US17/421,646 priority Critical patent/US20230138381A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

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  • the invention relates to the technical field of aloin, in particular to a method for LC-MS/MS determination of aloin in rat plasma.
  • Aloe vera is a chromoside substance, which exists in various plants of the Liliaceae Aloe genus, and the content in Aloe barbadensis Miller is about 3 ⁇ .
  • Aloe vera is a traditional Chinese medicine with a long history of application. The juice of its leaves is concentrated and dried to be used as medicine. Relevant studies have shown that aloe vera is one of the important active substances of aloe vera.
  • Aloe verain can inhibit the activity of tyrosinase, prevent the synthesis of melanin, and has a whitening effect.
  • Aloe can regulate the release of cellular inflammatory factors and growth factors to produce anti-inflammatory effects; induce the activation of Smad and MAPK signaling proteins to promote wound healing. Aloe can also activate the WNT signaling pathway, downregulate the Nortch signaling pathway, regulate the function of the digestive system, and improve the pathological state of colitis rats.
  • Aloe vera extract containing aloe verain can promote the secretion of adiponectin, regulate the metabolism of glucose and lipids in experimental animals, and have anti-diabetic effects.
  • Toxicological studies have shown that aloe verain basically has no toxic side effects.
  • the relevant reports do not provide pharmacokinetic parameters, and the analytical techniques used are relatively backward. The inventors seek to establish a more rapid and sensitive analysis method for aloe verain and apply it to pharmacokinetics research.
  • the present invention proposes an LC-MS/MS determination method for aloin in rat plasma, which can detect aloin in plasma more quickly, sensitively and accurately, and can be better applied to pharmacokinetic research.
  • a method for LC-MS/MS determination of aloin in rat plasma comprising the following steps:
  • Sample treatment of plasma take the plasma to be tested containing aloin, add methanol solution containing internal standard aloe veraside D, vortex precipitation, centrifuge, and take the supernatant as the sample to be tested;
  • the mass spectrometry conditions were set as follows: electrospray ion source, negative ion detection, spray voltage 4500V, spray temperature 550°C; multi-reaction ion monitoring scanning mode was adopted, and the ion pair used for quantitative analysis of aloe verain was m/z 393.1 ⁇ 272.9, the internal standard compound aloside D ion pair m/z is 555.3 ⁇ 144.9.
  • step (1) the methanol solution containing the internal standard aloside D and the plasma are mixed in a volume ratio of 2-4:1.
  • the concentration of the methanol solution containing the internal standard aloside D is 50 ng/mL to 100 ng/mL.
  • step (1) the rotating speed of the vortex is 2000rpm, and the time is 10min; the conditions of the centrifugation: 13000rpm, 4°C for 10min.
  • step (2) adopt filler to be the chromatographic column of C18, be a kind of in Synergi Hydro-Rp chromatographic column, Luna Omega C18 chromatographic column, Kinetex Evo C18 chromatographic column, and the specification of described chromatographic column is 2.10mm ⁇ 50mm, the particle size of the filler is 2-5 ⁇ m.
  • the chromatographic conditions the column temperature is 28-40° C., the injection volume is 1-10 ⁇ L, and the flow rate is 0.40-0.60 mL ⁇ min ⁇ 1 .
  • mass spectrometry conditions 30-100 psi for GS1, 30-100 psi for GS2, 20-50 psi for curtain gas, and 1-8 psi for collision gas.
  • the column temperature is 30° C.
  • the injection volume is 5 ⁇ L
  • the flow rate is 0.50 mL ⁇ min ⁇ 1 .
  • mass spectrometry conditions GS1 is 50 psi, GS2 is 50 psi, curtain gas is 30 psi, and collision gas is 2 psi.
  • the beneficial effects of the present invention are as follows: the present invention develops a LC-MS/MS determination method for aloe verain in rat plasma, which adopts a combination of liquid chromatography-mass spectrometer/mass spectrometer, and sets A specific gradient elution procedure, combined with certain mass spectrometry conditions, can rapidly, sensitively and accurately detect aloe verain in plasma, which can be better applied to pharmacokinetic studies.
  • Plasma samples (E) after 8 h of intravenous administration Plasma samples (E) after 8 h of intravenous administration.
  • Nexera XR UHPLC liquid chromatograph (Shimadzu Corporation, Japan) is connected to AB-SCIEX API 4000+ tandem quadrupole mass spectrometer through the Turbo V ion source interface, and Analyst software controls data acquisition and processing (American AB company).
  • Type 5922 refrigerated centrifuge (KUBOTA, Japan), XS105DU 1/100,000 electronic analytical balance (Mettler-Toledo, Switzerland), LabTower EDI15 ultra-pure water all-in-one machine (thermoelectric, USA), Vibrax circular oscillator (Aika, Germany) ).
  • AMS small animal anesthesia machine Beijing Ji Ander Technology Co., Ltd., Gene&I).
  • Aloesin reference substance was provided by Haorui Chemical (Shanghai) Co., Ltd.; Aloeresin D (Aloeresin D) reference substance was provided by Chengdu Purfield Biotechnology Co., Ltd., batch number: 19072605. The purity of the reference substance is all ⁇ 98%. Chromatographic grade methanol was provided by Merck, Germany. Ultrapure water was prepared by LabTower EDI15 ultrapure water machine. Isoflurane was provided by Beijing Jiandel Technology Co., Ltd.
  • Phase A is an aqueous solution containing 0.1 ⁇ (v/v) formic acid
  • mobile phase B is methanol
  • the gradient elution program is:
  • Electrospray ion source (ESI), negative ion detection, spray voltage 4500V, GS1 50psi, GS2 50psi, curtain gas 30psi, spray temperature 550°C, collision gas 2psi.
  • MRM Multiple reactive ion monitoring
  • the upper limit of quantification sample (600ng ⁇ mL-1) of the standard curve was taken for injection analysis, and then a blank sample was injected and analyzed to investigate the residues of aloe verain and internal standard aloeside D in the chromatogram of the blank sample.
  • the results showed that the residual peak area of aloe verain was less than 20% of the peak area of the lower limit of quantification sample, and the residual peak area of the internal standard aloside D was less than 5% of the internal standard peak area of the quality control sample, and the residual effect met the requirements.
  • aloe verain stock solution 0.1 mg ⁇ mL -1 of aloe verain stock solution to prepare low, medium and high concentrations (3.0, 50.0, 500.0 ng ⁇ mL -1 ) aloe verain control sample and quantitation lower limit sample (1.0 ng ⁇ mL -1 ).
  • ng ⁇ mL -1 5 parallel samples were set for each concentration, and after processing according to the method under "2.3", the samples were injected and analyzed according to the method under "2.1". The intra-day precision and accuracy were calculated, three batches of samples were measured within 3 days, and the inter-day precision was calculated.
  • the matrix effect was assessed by the post-extraction addition method, and the extraction recovery was also investigated.
  • High, medium and low concentrations (500ng ⁇ mL -1 , 50ng ⁇ mL -1 , 3ng ⁇ mL -1 ) of aloe verain samples were prepared with three different solvents, and five parallel samples were set for each concentration.
  • the sample solvent of Set2 series is blank plasma from different rats, and the supernatant is centrifuged after 3 times volume of methanol to precipitate protein;
  • the sample solvent of Set 3 series is blank plasma derived from different rats.
  • the ratio of aloin to the matrix factor of the internal standard aloside D was the normalized matrix factor. The results are shown in Table 2.
  • Example 1 The difference between this example and Example 1 is that the mobile phase A is 1.0 ⁇ (v/v) formic acid aqueous solution. After testing, the results showed that the peak shape of aloin and aloe glucoside D was good, and there was no mutual interference, and the endogenous substances in the plasma had no obvious effect on the signal of aloe verain and aloe glucoside D.
  • Example 1 The difference between this example and Example 1 is that the concentration of the methanol solution containing the internal standard aloside D is 100 ng/mL. After testing, the results showed that the peak height and peak area of aloe glucoside D increased by about 1 times, which can be used for the effective quantification of aloe verain.
  • Example 1 The difference between this example and Example 1 is that in the chromatographic conditions, the column temperature is 40°C. After testing, the results showed that the peak shape of aloin and aloe glucoside D was good, and there was no mutual interference, and the endogenous substances in the plasma had no obvious effect on the signal of aloe verain and aloe glucoside D.
  • Example 1 The difference between this example and Example 1 is that in the chromatographic conditions, the injection volume is 10 ⁇ L. After testing, the results showed that the peak shape of aloin and aloe glucoside D was good, and there was no mutual interference, and the endogenous substances in the plasma had no obvious effect on the signal of aloe verain and aloe glucoside D.
  • Example 1 The difference between this example and Example 1 is that in the chromatographic conditions, the chromatographic column is Luna Omega C18 (2.10mm ⁇ 50mm, 4 ⁇ m, phenomenex). After testing, the results showed that the peak shape of aloin and aloe glucoside D was good, and there was no mutual interference, and the endogenous substances in the plasma had no obvious effect on the signal of aloe verain and aloe glucoside D.
  • Example 1 The difference between this example and Example 1 is that in the chromatographic conditions, the chromatographic column is Kinetex Evo C18 (2.10mm ⁇ 50mm, 4 ⁇ m, phenomenex). After testing, the results showed that the peak shape of aloin and aloe glucoside D was good, and there was no mutual interference, and the endogenous substances in the plasma had no obvious effect on the signal of aloe verain and aloe glucoside D.
  • Example 1 The difference between this example and Example 1 is that, in the mass spectrometry conditions, GS1 is 100 psi, GS2 is 100 psi, curtain gas is 50 psi, and collision gas is 8 psi. After testing, the results showed that the peak heights of aloin and aloside D were slightly reduced, and the signal-to-noise ratio (S/N) of the sample at the lowest limit of quantification was ⁇ 10.
  • step (2) the gradient elution procedure is:
  • the results show that the retention time of aloin is about 1min, and there is a strong impurity peak interference signal near the peak position of the chromatographic peak, so that the lower limit of quantification of aloin in plasma is less than 1ng/mL.
  • step (3) the mass spectrometry conditions are set as: electrospray ion source, negative ion detection, spray voltage 5000V, spray temperature 500°C;
  • the ion pair for quantitative analysis of aloe verain was m/z 393.1 ⁇ 272.9, and the m/z of the internal standard compound aloside D was 555.25 ⁇ 511.2.
  • the results showed that the signal intensity of aloin and aloside D was 2 orders of magnitude lower than that in the positive ion mode.

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Abstract

一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，包括以下步骤：(1)血浆的样品处理：取含芦荟苦素的待测血浆，加入含内标芦荟新苷D的甲醇溶液涡旋沉淀，离心，取上清液为待检测样品；(2)设定液相色谱条件为：采用流动相A和流动相B进行梯度洗脱，流动相A为含0.1～1.0‰(v/v)甲酸水溶液，流动相B为甲醇；(3)设定质谱条件为：电喷雾离子源，负离子检测，喷雾电压4500V，喷雾温度为550℃，采用多反应离子监测扫描模式，用于芦荟苦素定量分析的离子对为m/z 393.1→272.9，内标化合物芦荟新苷D离子对m/z为555.3→144.9；该方法可更加快速、灵敏、准确地检测血浆中芦荟苦素。

Description

一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法 技术领域
本发明涉及芦荟苦素技术领域，特别涉及一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法。
背景技术
芦荟苦素是一种色酮苷类物质，在百合科芦荟属多种植物中均有存在，在芦荟Aloe barbadensis Miller中的含量约为3‰左右。芦荟是一种应用历史悠久的传统中药，用其叶的汁液浓缩干燥后入药，具有泻下通便，清肝泻火，杀虫疗疳之功效。相关研究显示，芦荟苦素是芦荟的重要活性物质之一。芦荟苦素可以抑制酪氨酸酶活性，阻止黑色素的合成，具有美白功效。芦荟苦素可以调节细胞炎症因子和生长因子释放，产生抗炎作用；诱导Smad和MAPK信号蛋白的激活，促进伤口愈合。芦荟苦素还可以激活WNT信号通路，下调Nortch信号通路，调节消化***功能，改善结肠炎大鼠病理状态。
含芦荟苦素的芦荟提取物可以促进脂联素的分泌，调节实验动物糖脂代谢，有抗糖尿病作用。毒理学研究表明，芦荟苦素基本无毒副作用。然而，芦荟苦素的药代动力学研究很少，有关报道没有提供药代动力学参数，所用分析技术也已相对落后。本发明人力求建立一种更加快速、灵敏的芦荟苦素分析方法，并应用于药动学研究。
发明内容
鉴于此，本发明提出一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，可更加快速、灵敏、准确地检测血浆中芦荟苦素，可更好地应用于药动学研究。
本发明的技术方案是这样实现的：
一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，包括以下步骤：
(1)血浆的样品处理：取含芦荟苦素的待测血浆，加入含内标芦荟新苷D的甲醇溶液涡旋沉淀，离心，取上清液为待检测样品；
(2)设定液相色谱条件为：采用流动相A和流动相B进行梯度洗脱，流动相A为含0.1～1.0‰(v/v)甲酸水溶液，流动相B为甲醇，梯度洗脱程序为：
时间/min 流动相A/％ 流动相B/％
0～0.50 95 5
0.51～3.00 95～5 5～95
3.01～4.00 10 90
4.01～5.00 95 5
(3)设定质谱条件为：电喷雾离子源，负离子检测，喷雾电压4500V，喷雾温度为550℃；采用多反应离子监测扫描模式，用于芦荟苦素定量分析的离子对为m/z 393.1→272.9，内标化合物芦荟新苷D离子对m/z为555.3→144.9。
进一步的，步骤(1)中，所述含内标芦荟新苷D的甲醇溶液与血浆按体积比2-4：1进行混合。
进一步的，步骤(1)中，所述含内标芦荟新苷D的甲醇溶液的浓度为50ng/mL～100ng/mL。
进一步的，步骤(1)中，所述涡旋的转速为2000rpm，时间为10min；所述离心的条件：13 000rpm、4℃离心10min。
进一步的，步骤(2)中，采用填料为C18的色谱柱，为Synergi Hydro-Rp色谱柱、Luna Omega C18色谱柱、Kinetex Evo C18色谱柱中的一种，所述色谱柱的规格为2.10mm×50mm，填充剂粒径为2-5μm。
进一步的，色谱条件：柱温28～40℃，进样量1～10μL，流速0.40～0.60mL·min -1。
进一步的，质谱条件：GS1为30-100psi，GS2为30-100psi，帘气为20-50psi，碰撞气为1-8psi。
进一步的，所述多反应离子监测的参数为：
  DP/V CE/V CXP/V EP/V
芦荟苦素 90 29 10 10
芦荟新苷D 115 41 11 10
。
进一步的，所述柱温为30℃，进样量为5μL，流速为0.50mL·min -1。
进一步的，质谱条件：GS1为50psi，GS2为50psi，帘气为30psi，碰撞气为 2psi。
与现有技术相比，本发明的有益效果是：本发明开发出一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，采用液相色谱-质谱/质谱仪联用，设定特定的梯度洗脱程序，结合一定的质谱条件，可快速、灵敏、准确地检测血浆中芦荟苦素，可更好地应用于药动学研究。
附图说明
图1芦荟苦素与芦荟新苷D的化学结构与MS/MS谱图。
图2芦荟苦素和内标芦荟新苷D的LC-MS/MS色谱图，
空白血浆样品(不加内标处理，A)、
标准曲线定量下限样品(芦荟苦素1ng·mL -1，B)、
空白血浆样品(加内标处理，C)、口服给药8h后血浆样品(D)、
静脉给药8h后血浆样品(E)。
具体实施方式
为了更好理解本发明技术内容，下面提供具体实施例，对本发明做进一步的说明。
本发明实施例所用的实验方法如无特殊说明，均为常规方法。
本发明实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
1仪器、试药与实验动物
1.1仪器
Nexera XR UHPLC液相色谱仪(日本岛津公司)通过Turbo V离子源接口串联AB-SCIEX API 4000+串联四级杆质谱仪，Analyst软件控制数据采集和处理(美国AB公司)。5922型冷冻离心机(日本KUBOTA)，XS105DU十万分之一电子分析天平(瑞士梅特勒-托利多公司)，LabTower EDI15超纯水一体机(美国热电)，Vibrax圆周振荡器(德国艾卡)。AMS小动物麻醉机(北京吉 安得尔科技有限公司，Gene&I)。
1.2试药与实验动物
芦荟苦素对照品由昊睿化学(上海)有限公司提供；芦荟新苷D(Aloeresin D)对照品由成都普菲德生物技术有限公司提供，批号：19072605。对照品纯度均≥98％。色谱级甲醇由德国默克公司提供。超纯水由LabTower EDI15超纯水机制备。异氟烷由北京吉安得尔科技有限公司提供。
清洁级SD雄性大鼠，体重243～300g(长沙市天勤生物技术有限公司提供，实验动物合格证号：No.43006700017619，许可证号：SCXK(湘)2014-0011)；饲养条件：温度22±2℃，湿度40％～70％，12h明暗交替，自由进食、饮水。
2方法与结果
2.1色谱-质谱条件
采用Synergi Hydro-Rp(2.10mm×50mm,4μm,phenomenex)色谱柱，柱温30℃，流速0.50mL·min -1,进样量5μL，采用流动相A和流动相B进行梯度洗脱，流动相A为含0.1‰(v/v)甲酸水溶液，流动相B为甲醇，梯度洗脱程序为：
时间/min 流动相A/％ 流动相B/％
0～0.50 95 5
0.51～3.00 95～5 5～95
3.01～4.00 10 90
4.01～5.00 95 5
。
电喷雾离子源(ESI)，负离子检测，喷雾电压4500伏，GS1为50psi，GS2为50psi，帘气为30psi，喷雾温度为550℃，碰撞气为2psi。
多反应离子监测(MRM)，用于芦荟苦素定量分析的离子对为m/z 393.1→272.9(DP:-90,CE:-29,CXP:-10,EP:-10),内标化合物芦荟新苷D为555.3→144.9(DP:-115,CE:-41,CXP:-11,EP:-10)。
芦荟苦素与芦荟新苷D的化学结构与质谱图如下图1.
2.2溶液制备
精密称取芦荟苦素对照品适量，加甲醇配制成0.1mg·mL -1的储备液。取芦荟新苷D对照品适量，精密称定，加甲醇配制成50ng·mL -1内标溶液。配制好的溶液置4℃保存备用。
2.3血浆样品处理
取血浆50μL置离心管中，加入含内标芦荟新苷D 50ng·mL -1的甲醇溶液150μL，2000rpm涡旋10min充分沉淀蛋白，13 000rpm(4℃)离心10min，取上清液置微量进样管中，进样5μL进行LC-MS/MS分析。
2.4方法学考察
按2020年版《中国药典》四部生物样品定量分析方法验证指导原则的要求，验证分析方法的有效性。
2.4.1专属性考察
取来源不同大鼠的空白血浆、空白血浆加芦荟苦素对照品、静脉给药后8h经眼眶静脉采集到的血浆样品、口服给药8h后血浆样品按“2.3”项下样品处理方法处理，按“2.1”项下条件进样分析，色谱图见图2。芦荟苦素、芦荟新苷D出峰时间分别为1.85、2.67min。
2.4.2线性关系考察
取空白血浆，加入0.1mg·mL -1的芦荟苦素储备液配制成600、400、200、40、10、2、1ng·mL -1系列浓度的标准曲线血浆样品，各取50μL经“2.3”项下方法处理后，进样5μL按“2.1”项下方法进行分析。以待测物与内标峰面积之比(Y)为纵坐标，待测物浓度(X)为横坐标，权重为1/X 2，进行线性回归，得回归方程Y＝0.00617X+0.00113(r＝0.9945)。表明芦荟苦素在1～600ng·mL -1范围内线性 关系良好。
2.4.3残留效应
取标准曲线定量上限样品(600ng·mL-1)处理进样分析后，接着进样分析1个空白样品，考察分析芦荟苦素和内标芦荟新苷D在空白样品色谱图中的残留。结果，芦荟苦素残留的峰面积小于定量下限样品峰面积的20％，内标芦荟新苷D的残留峰面积小于质控样品内标峰面积的5％，残留效应符合要求。
2.4.4准确度与精密度
取空白血浆，加入0.1mg·mL -1的芦荟苦素储备液配制成低、中、高三种浓度(3.0、50.0、500.0ng·mL -1)的芦荟苦素质控样品和定量下限样品(1.0ng·mL -1)，每个浓度设5个平行样，按“2.3”项下方法处理后，按“2.1”项下方法进样分析。计算日内精密度和准确度，3d内测定3批样品，计算日间精密度。
结果日内精密度、日间精密度RSD均小于15％，重复性良好；准确度在96.96％～107.28％之间。具体结果见表1.
表1芦荟苦素定量分析精密度和准确度结果
Figure PCTCN2021080459-appb-000001
2.4.5基质效应和提取回收率试验
按现有文献方法，采用提取后添加法评定基质效应，同时考察提取回收率。用3种不同溶剂分别配制高、中、低3种浓度(500ng·mL -1、50ng·mL -1、3ng·mL -1)的芦荟苦素样品，每个浓度设5个平行样。
Set 1系列样品溶剂为甲醇；
Set2系列样品溶剂为来源于不同大鼠的空白血浆，经3倍体积甲醇沉淀蛋 白后，离心分取的上清液；
Set 3系列样品溶剂为来源于不同大鼠的空白血浆。
各取以上样品50μL，按“2.3”项下方法处理后，按“2.1”项下方法进样分析，得到峰面积为分别为A 1、A 2、A 3，基质因子MF＝A 2/A 1×100％；提取回收率RE＝A 3/A 2×100％。
芦荟苦素与内标芦荟新苷D的基质因子的比值为归一化的基质因子。结果见表2.
表2大鼠血浆中芦荟苦素的基质效应和提取回收率(Mean±SD,n＝5)
Figure PCTCN2021080459-appb-000002
2.4.6稳定性考察
用空白血浆配制3种浓度(500ng·mL -1、50ng·mL -1、3ng·mL -1)的芦荟苦素质控样品，考察下述情况下的稳定性。
(1)样品处理好后放置自动进样器中8h后进样分析；
(2)样品经3个冻融循环后再处理，进样分析；
(3)样品在室温下放置4h后再出来，进样分析；
(4)样品在-20℃下保存15d后再处理，进样分析。
结果各浓度样品测试的准确度在94.66％～109.60％之间，RSD均小于15％。稳定性考察合格。结果见表3.
表3血浆样品中芦荟苦素的稳定性
Figure PCTCN2021080459-appb-000003
实施例2
本实施例与实施例1的区别在于，流动相A为1.0‰(v/v)甲酸水溶液。经检测，结果显示芦荟苦素与芦荟新苷D峰形良好，无相互干扰，血浆中内源性物质对芦荟苦素和芦荟新苷D信号无明显影响。
实施例3
本实施例与实施例1的区别在于，含内标芦荟新苷D的甲醇溶液的浓度为100ng/mL。经检测，结果显示芦荟新苷D峰高、峰面积增加约1倍，可以用于芦荟苦素的有效定量。
实施例4
本实施例与实施例1的区别在于，色谱条件中，柱温40℃。经检测，结果显示芦荟苦素与芦荟新苷D峰形良好，无相互干扰，血浆中内源性物质对芦荟苦素和芦荟新苷D信号无明显影响。
实施例5
本实施例与实施例1的区别在于，色谱条件中，进样量10μL。经检测，结果显示芦荟苦素与芦荟新苷D峰形良好，无相互干扰，血浆中内源性物质对芦荟苦素和芦荟新苷D信号无明显影响。
实施例6
本实施例与实施例1的区别在于，色谱条件中，色谱柱为Luna Omega C18(2.10mm×50mm,4μm,phenomenex)。经检测，结果显示芦荟苦素与芦荟新苷D峰形良好，无相互干扰，血浆中内源性物质对芦荟苦素和芦荟新苷D信号无明显影响。
实施例7
本实施例与实施例1的区别在于，色谱条件中，色谱柱为Kinetex Evo  C18(2.10mm×50mm,4μm,phenomenex)。经检测，结果显示芦荟苦素与芦荟新苷D峰形良好，无相互干扰，血浆中内源性物质对芦荟苦素和芦荟新苷D信号无明显影响。
实施例8
本实施例与实施例1的区别在于，质谱条件中，GS1为100psi，GS2为100psi，帘气为50psi，碰撞气为8psi。经检测，结果显示芦荟苦素与芦荟新苷D峰高略有降低，最低定量限样品的信噪比(S/N)≥10。
对比例1
本对比例与实施例1的区别在于，步骤(2)中，梯度洗脱程序为：
时间/min 流动相A/％ 流动相B/％
0～1.0 80 20
1.01～2.50 50 50
2.51～3.50 20 80
4.01～5.00 90 10
经检测，结果显示芦荟苦素保留时间约1min，色谱峰出峰位置附近有较强的杂质峰干扰信号，使血浆中芦荟苦素的定量下限达不到1ng/mL.
对比例2
本对比例与实施例1的区别在于，步骤(3)中，设定质谱条件为：电喷雾离子源，负离子检测，喷雾电压5000V，喷雾温度为500℃；采用多反应离子监测扫描模式，用于芦荟苦素定量分析的离子对为m/z 393.1→272.9，内标化合物芦荟新苷D离子对m/z为555.25→511.2。结果显示芦荟苦素与芦荟新苷D信号强度比正离子模式下低2个数量级。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，其特征在于，包括以下步骤：
    (1)血浆的样品处理：取含芦荟苦素的待测血浆，加入含内标芦荟新苷D的甲醇溶液涡旋沉淀，离心，取上清液为待检测样品；
    (2)设定液相色谱条件为：采用流动相A和流动相B进行梯度洗脱，流动相A为含0.1～1.0‰(v/v)甲酸水溶液，流动相B为甲醇，梯度洗脱程序为：
    时间/min 流动相A/％ 流动相B/％ 0～0.50 95 5 0.51～3.00 95～5 5～95 3.01～4.00 10 90 4.01～5.00 95 5
    ；
    (3)设定质谱条件为：电喷雾离子源，负离子检测，喷雾电压4500V，喷雾温度为550℃；采用多反应离子监测扫描模式，用于芦荟苦素定量分析的离子对为m/z 393.1→272.9，内标化合物芦荟新苷D离子对m/z为555.3→144.9。
  2. 根据权利要求1所述的一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，其特征在于，步骤(1)中，所述含内标芦荟新苷D的甲醇溶液与血浆按体积比2-4：1进行混合。
  3. 根据权利要求1所述的一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，其特征在于，步骤(1)中，所述含内标芦荟新苷D的甲醇溶液的浓度为50ng/mL～100ng/mL。
  4. 根据权利要求1所述的一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，其特征在于，步骤(1)中，所述涡旋的转速为2000rpm，时间为10min；所述离心的条件：13000rpm、4℃离心10min。
  5. 根据权利要求1所述的一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，其特征在于，步骤(2)中，采用填料为C18的色谱柱，为Synergi Hydro-Rp色谱柱、Luna Omega C18色谱柱、Kinetex Evo C18色谱柱中的一种，所述色谱柱的规格为2.10mm×50mm，填充剂粒径为2-5μm。
  6. 根据权利要求1所述的一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，其特征在于，色谱条件：柱温28～40℃，进样量1～10μL，流速0.40～0.60mL·min -1。
  7. 根据权利要求1所述的一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，其特征在于，质谱条件：GS1为30-100psi，GS2为30-100psi，帘气为20-50psi，碰撞气为1-8psi。
  8. 根据权利要求1所述的一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，其特征在于，所述多反应离子监测的参数为：
      DP/V CE/V CXP/V EP/V 芦荟苦素 90 29 10 10 芦荟新苷D 115 41 11 10
    。
  9. 根据权利要求6所述的一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，其特征在于，所述柱温为30℃，进样量为5μL，流速为0.50mL·min -1。
  10. 根据权利要求7所述的一种大鼠血浆中芦荟苦素的LC-MS/MS测定方法，其特征在于，质谱条件：GS1为50psi，GS2为50psi，帘气为30psi，碰撞气为2psi。
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