CN112198269A - 一种测定Beagle犬血浆中羟基酪醇的方法 - Google Patents
一种测定Beagle犬血浆中羟基酪醇的方法 Download PDFInfo
- Publication number
- CN112198269A CN112198269A CN202010321750.4A CN202010321750A CN112198269A CN 112198269 A CN112198269 A CN 112198269A CN 202010321750 A CN202010321750 A CN 202010321750A CN 112198269 A CN112198269 A CN 112198269A
- Authority
- CN
- China
- Prior art keywords
- hydroxytyrosol
- plasma
- sample
- determining
- beagle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- JUUBCHWRXWPFFH-UHFFFAOYSA-N Hydroxytyrosol Chemical compound OCCC1=CC=C(O)C(O)=C1 JUUBCHWRXWPFFH-UHFFFAOYSA-N 0.000 title claims abstract description 170
- 235000003248 hydroxytyrosol Nutrition 0.000 title claims abstract description 84
- 229940095066 hydroxytyrosol Drugs 0.000 title claims abstract description 84
- 238000000034 method Methods 0.000 title claims abstract description 55
- 241000282472 Canis lupus familiaris Species 0.000 title claims abstract description 43
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 57
- JLRGJRBPOGGCBT-UHFFFAOYSA-N Tolbutamide Chemical compound CCCCNC(=O)NS(=O)(=O)C1=CC=C(C)C=C1 JLRGJRBPOGGCBT-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229960005371 tolbutamide Drugs 0.000 claims abstract description 17
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 238000004885 tandem mass spectrometry Methods 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 238000000132 electrospray ionisation Methods 0.000 claims abstract description 3
- 210000002381 plasma Anatomy 0.000 claims description 71
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 24
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 19
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 12
- 235000019253 formic acid Nutrition 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 239000012086 standard solution Substances 0.000 claims description 10
- 239000012071 phase Substances 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 7
- 239000005695 Ammonium acetate Substances 0.000 claims description 7
- 235000019257 ammonium acetate Nutrition 0.000 claims description 7
- 229940043376 ammonium acetate Drugs 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 239000007791 liquid phase Substances 0.000 claims description 5
- 238000001819 mass spectrum Methods 0.000 claims description 3
- 239000003595 mist Substances 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 50
- 238000004458 analytical method Methods 0.000 abstract description 16
- 239000011159 matrix material Substances 0.000 abstract description 14
- 238000011160 research Methods 0.000 abstract description 13
- 239000000126 substance Substances 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 8
- 238000000605 extraction Methods 0.000 abstract description 6
- 239000012472 biological sample Substances 0.000 abstract description 5
- 238000011084 recovery Methods 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 230000006920 protein precipitation Effects 0.000 abstract description 3
- 150000002500 ions Chemical class 0.000 description 11
- 238000003908 quality control method Methods 0.000 description 9
- 238000010253 intravenous injection Methods 0.000 description 7
- 239000013582 standard series solution Substances 0.000 description 7
- 239000011550 stock solution Substances 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- 241000282465 Canis Species 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000004006 olive oil Substances 0.000 description 3
- 235000008390 olive oil Nutrition 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 235000020927 12-h fasting Nutrition 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000233805 Phoenix Species 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 238000011953 bioanalysis Methods 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 210000003194 forelimb Anatomy 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 238000000611 regression analysis Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- RFWGABANNQMHMZ-UHFFFAOYSA-N 8-acetoxy-7-acetyl-6,7,7a,8-tetrahydro-5H-benzo[g][1,3]dioxolo[4',5':4,5]benzo[1,2,3-de]quinoline Natural products CC=C1C(CC(=O)OCCC=2C=C(O)C(O)=CC=2)C(C(=O)OC)=COC1OC1OC(CO)C(O)C(O)C1O RFWGABANNQMHMZ-UHFFFAOYSA-N 0.000 description 1
- HKVGJQVJNQRJPO-UHFFFAOYSA-N Demethyloleuropein Natural products O1C=C(C(O)=O)C(CC(=O)OCCC=2C=C(O)C(O)=CC=2)C(=CC)C1OC1OC(CO)C(O)C(O)C1O HKVGJQVJNQRJPO-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- CNIUEVQJABPUIJ-QMMMGPOBSA-N N-hydroxytyrosine Chemical compound ON[C@H](C(O)=O)CC1=CC=C(O)C=C1 CNIUEVQJABPUIJ-QMMMGPOBSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 235000002725 Olea europaea Nutrition 0.000 description 1
- RFWGABANNQMHMZ-HYYSZPHDSA-N Oleuropein Chemical compound O([C@@H]1OC=C([C@H](C1=CC)CC(=O)OCCC=1C=C(O)C(O)=CC=1)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RFWGABANNQMHMZ-HYYSZPHDSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229930182489 iridoid glycoside Natural products 0.000 description 1
- 150000008145 iridoid glycosides Chemical class 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 235000011576 oleuropein Nutrition 0.000 description 1
- RFWGABANNQMHMZ-CARRXEGNSA-N oleuropein Natural products COC(=O)C1=CO[C@@H](O[C@H]2O[C@@H](CO)[C@H](O)[C@@H](O)[C@@H]2O)C(=CC)[C@H]1CC(=O)OCCc3ccc(O)c(O)c3 RFWGABANNQMHMZ-CARRXEGNSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
本发明涉及一种测定Beagle犬血浆中羟基酪醇的方法,属于生物检测领域。该方法采用甲苯磺丁脲为内标,乙腈沉淀蛋白处理后,采用高效液相色谱法‑电喷雾离子化串联质谱法来检测羟基酪醇在Beagle犬血浆样品中的浓度。本发明方法在准确度、精密度、专属性、稳定性、提取回收率及基质效应等方面均达到了中国药典2015年版以及NMPA2014年颁布的《化学药物非临床药代动力学研究技术指导原则》对生物样品分析的要求。
Description
技术领域
本发明涉及一种测定Beagle犬血浆中羟基酪醇的方法,属于生物检测领域。
背景技术
羟基酪醇(hydroxytyrosol,HT),化学名称为3,4-二羟基苯乙醇,是存在于橄榄叶和橄榄油中的天然多酚类成分,具有体外抗氧化作用,常用于保健品和食品添加剂。最近研究表明,羟基酪醇具有抗癌、抗菌、抗炎、抗氧化、保护心脏等多种有益于人体健康的药理活性,其药用价值亟待开发,对羟基酪醇的成药性研究和临床研究将是未来主要研究方向。而对羟基酪醇成药性评价的临床前研究涉及不同种属实验动物的药代/毒代动力学研究,因此针对各类生物样本建立快速、灵敏、专属性强的羟基酪醇分析检测方法十分必要。
目前关于羟基酪醇的体内分析方法较少,针对不同生物基质开发的分析方法也不相同。例如文献1(杨茜.LC-MS/MS法研究羟基酪醇对甲苯磺酸酯药代动力学及组织分布[D],唐山:华北理工大学,2019.)公开了一种测定大鼠血浆及组织中羟基酪醇的LC-MS/MS方法并成功应用于羟基酪醇前体药物大鼠体内药代动力学及组织分布研究;文献2(PieroDel Boccio,Antonietta Di Deo,Amalia De Curtis,etal.Liquid chromatography-tandem mass spectrometry analysis of oleuropein and its metabolitehydroxytyrosol in rat plasma and urine after oral administration[J].JChromatogr B Analyt Technol Biomed Life Sci,2003,785(1):47-56.)公开了LC-MS/MS测定大鼠血浆及尿液中环烯醚萜苷及其生物活性代谢物羟基酪氨酸的定量方法;此外对于人服用橄榄油后血浆中羟基酪醇的含量测定也有相关报道(Pastor A,Rodríguez-Morató,Jose,etal.Analysis of free hydroxytyrosol in human plasma following theadministration ofolive oil[J].Journal ofChromatographyA,2016:183-190.)。而对于Beagle犬血浆中羟基酪醇的含量测定方法尚未见文献报道,由于羟基酪醇极易被氧化,稳定性差,药代动力学研究要求的生物分析方法必须灵敏准确,本发明首次建立了利用LC/MS/MS测定Beagle犬血浆中羟基酪醇的检测方法,为羟基酪醇的临床前研究奠定了坚实的生物分析基础。
发明内容
本发明的目的在于提供一种Beagle犬血浆中羟基酪醇的检测方法。
本发明的测定Beagle犬血浆中羟基酪醇的方法,其包括采用高效液相色谱法-电喷雾离子化串联质谱法即HPLC-MS-MS法来检测Beagle犬血浆中羟基酪醇的浓度。
本发明所述的测定Beagle犬血浆中羟基酪醇的方法,其具体包括如下步骤:
(1)血浆样品处理:100μL血浆样品置于1.5mL离心管中,加入10μL甲苯磺丁脲内标溶液及500μL乙腈溶液,涡旋2min,13000rpm(8℃)离心5min,取上清液作为待测液;
(2)取上述待测液1μL进行LC-MS/MS分析,其中液相系统条件为:A相为0.1%甲酸+5mM醋酸铵水,B相为乙腈(含0.1%甲酸),梯度洗脱:0~0.7min 2%B,0.7~1.5min2%→90%B,1.6~2.5min 2%B;色谱柱为Accucore aQ C18色谱柱(2.1×50mm,2.6μm);流速:0.5mL/min;柱温:40℃;进样室温度:15℃;进样量:1.0μL;样品检测时间:2.5min。
质谱系统条件为:AB SCIEX5500三重四级杆串联质谱系统;离子源:ESI源;负离子模式;气帘气压力:-35psi;碰撞气:-8psi;离子化电压:-5500psi;离子源温度:550℃;喷雾气:-55psi;辅助加热气:-55psi;多重反应监测(MRM)模式。
(3)羟基酪醇标准曲线的测定:取Beagle犬空白血浆,加入羟基酪醇标准系列溶液,配制成相当于羟基酪醇浓度为1,2,5,10,50,200,500,1000,2000ng/mL的血浆标准样品,按步骤(1)、步骤(2)所述的测定方法操作,记录色谱图;以待测物浓度为横坐标,待测物与内标物的峰面积比值为纵坐标,用加权最小二乘法(权重为1/x2)进行回归分析,确定羟基酪醇的血浆样品标准曲线及其定量限。
(4)羟基酪醇在Beagle犬体内药代动力学试验:采用两周期交叉试验设计。4只Beagle犬,雄性,随机分成2组。禁食12h后,静注组静脉注射给予羟基酪醇溶液,口服组口服给予羟基酪醇胶囊,给药剂量14mg/kg。各组于给药后0.083、0.167、0.333、0.5、1、2、4、6、8和24h自犬前肢静脉取血约1.5mL。
采集后置于冰上,并于1h之内离心分离血浆(离心条件:8000rpm,10min,2-8℃)。14天后,两组动物交叉给药,按上述方法取静脉血并制备血浆。收集的血浆按照步骤(1)中的方法预处理并按照步骤(2)中的高效液相色谱-串联质谱(LC-MS/MS)的高通量测定方法以及步骤(3)中所获得羟基酪醇标准曲线计算确定不同取样时间的血浆中羟基酪醇的浓度,并利用Phoenix WinNonlin8.1软件确定羟基酪醇的体内药代动力学参数。
血浆样品的测定前预处理是保证LC-MS/MS法灵敏度的关键过程。常见的处理方法包括蛋白沉淀法、固相萃取法和液-液萃取法。本方法采用操作相对简单的蛋白沉淀法,采用乙腈作为沉淀溶剂,峰形好且能保证灵敏度。
液相条件中流动相由0.1%甲酸+5mM醋酸铵水和含0.1%甲酸的乙腈组成,梯度洗脱,流动相酸化后可保证羟基酪醇出峰稳定;由于羟基酪醇极性较大,采用Accucore aQC1850mm短柱进行分析,峰型良好,单个样品的分析时间仅为2.5min,有利于大批量样品的快速分析。
母离子/子离子离子对的选择是建立本发明中检测方法的关键。样品直接以恒定流速由微量注射泵流入质谱仪时,负离子模式一级全扫描可见明显的[M-H]-母离子峰,m/z为152.9,二级碎片离子峰度最大的为m/z为123.0。改变CE和DP的大小可明显影响峰强度,通过优化,确定本方法最优的CE为-20V,DP为-70V。
内标的选择对于检测的灵敏度和特异性至关重要,理想的内标应为同位素标记物或结构类似物。申请人在工作开展中发现,甲苯磺丁脲的LC-MS/MS检出条件与羟基酪醇类似,并且预实验发现两者在此条件下出峰时间相近、无相互干扰,峰形均好,因此本方法确定将甲苯磺丁脲作为羟基酪醇Beagle血浆生物样品测定的内标。
本发明所建立的测定Beagle犬血浆中羟基酪醇的方法,在准确度、精密度、专属性、稳定性、提取回收率及基质效应等方面均达到了中国药典2015年版以及NMPA2014年颁布的《化学药物非临床药代动力学研究技术指导原则》对生物样品的分析要求。
附图说明
图1Beagle犬空白血浆色谱图(不同Beagle犬)
图2Beagle犬空白血浆中加入羟基酪醇及内标处理后检测得到的色谱图
图3Beagle犬血浆羟基酪醇的标准曲线图
具体实施方式
为了使本领域技术人员更清楚地了解本发明,申请人对本发明所述一种测定Beagle犬血浆中羟基酪醇的方法进一步描述如下,其中检测方法的方法学考查以Beagle犬血浆样本为例,具体实施以羟基酪醇在Beagle犬体内药代动力学试验为例,但本领域技术人员应能知晓,所述的进一步阐述和举例并不以任何方式限定本发明的专利保护范围。
实施例1本发明检测方法的方法学考察
一、标准溶液的制备
(1)标准系列溶液的配制
精密称定羟基酪醇适量,加入溶媒(含0.1%甲酸,5mM醋酸铵,5mM抗坏血酸水溶液)并定量稀释制成4mg/mL的标准储备液。精密量取适量标准储备液,用溶媒梯度稀释,配制浓度分别为10,20,50,200,500,2000,5000,10000,20000ng/mL的标准系列溶液。
(2)内标溶液的配制
精密称定甲苯磺丁脲适量,加乙腈溶解并定量稀释制成10mg/mL的甲苯磺丁脲储备液。精密量取适量储备液,用乙腈逐步稀释得浓度为200ng/mL的内标乙腈溶液。
(3)标准质控溶液的配制
精密称定羟基酪醇适量,加入溶媒溶解并定量稀释制成1.0mg/mL的标准质控储备液。精密量取适量标准质控储备液,用80%乙腈梯度稀释,配制浓度分别为20,2000,10000ng/mL的标准系列溶液。
羟基酪醇储备液及标准系列溶液现用现配,内标溶液置4℃冰箱保存备用。
二、分析方法的确证
(1)血浆样品预处理
取100μL血浆样品置于1.5mL离心管中,加入10μL甲苯磺丁脲内标溶液及500μL乙腈溶液,涡旋2min,13000rpm(8℃)离心5min,取上清液1μL进行LC-MS/MS分析。
(2)方法的专属性
取6个不同来源Beagle犬空白血浆各100μL,除用乙腈代替内标溶液外,其余按“血浆样品预处理”项下方法操作,获得空白基质色谱图,如图1所示;将一定浓度的羟基酪醇对照品溶液和内标溶液加入空白血浆溶液中,依法操作,获得相应的色谱图,如图2所示。其中羟基酪醇的保留时间为0.68min,甲苯磺丁脲的保留时间为1.02min;结果表明,Beagle犬血浆中内源性物质不干扰测定。
(3)标准曲线和线性范围
取Beagle犬空白血浆,依次加入羟基酪醇标准系列溶液,配制成相当于羟基酪醇浓度为1,2,5,20,50,200,500,10000,2000的模拟血浆样品,按“血浆样品预处理”项下操作。以待测物浓度为横坐标,待测物与内标物的峰面积比值为纵坐标,用加权最小二乘法(权重为1/x2)进行回归分析。典型标准曲线为Y=0.0042X+0.0646,r=0.9931;定量下限(LLOQ)为1ng/mL。
(4)精密度和准确度
取Beagle犬空白血浆,加入羟基酪醇标准系列溶液,配制低(2ng/mL)、中(200ng/mL)、高(1000ng/mL)三个浓度的质量控制(QC)样品。按“血浆样品预处理”项下操作,每一浓度进行6样本分析,连续测定3天,随行标准曲线。根据当日标准曲线计算QC样品的浓度,结果进行方差分析,求得方法的精密度RSD和准确度RE,羟基酪醇的日间RSD≤10%,日内≤13%,RE在-13.0%~9.7%之间。
(5)基质效应
使用6个不同来源的Beagle犬空白血浆,配制低(2ng/mL)、中(200ng/mL)、高(1000ng/mL)三个浓度水平进行基质效应考察。
样品A:取Beagle犬空白血浆100μL,加入500μL乙腈,涡旋2min,8℃,13000r/min离心5min,作为提取后空白基质;取以上上清液,加入羟基酪醇质控工作液及甲苯磺丁脲后再次涡旋离心,取上清液进样分析。
样品B:取纯化水,直接加入羟基酪醇质控工作液和甲苯磺丁脲溶液,涡旋离心,取上清液进样分析。
通过计算样品A的峰面积与样品B的相应峰面积比值,计算分析物和内标的基质因子。进一步通过分析物的基质因子除以内标的基质因子,计算经内标归一化的基质因子,考察基质效应。
结果表明羟基酪醇低、中、高浓度的基质效应变异系数均小于15%。因此本试验选择的色谱和质谱条件下,可忽略基质效应对羟基酪醇测定的影响。
(6)提取回收率
样品C:配制高、中、低浓度水平的质控样品,采用“血浆样品预处理”的方法进行样品处理,取上清液进样分析。
样品C测得的羟基酪醇峰面积与相应浓度的样品A测得峰面积比即为羟基酪醇的提取回收率。
结果表明羟基酪醇低、中、高三个浓度的提取回收率分别为85.6%,89.6%和87.7%。
(7)样品稳定性
本专利考察了未处理的血浆样品室温放置4h的稳定性,处理后进样室(15℃)放置6h的稳定性,血浆样品经过3次冷冻(-80℃)-解冻循环的稳定性,血浆样品长期冷冻(-80℃)放置10d的稳定性。稳定性考察时,每一浓度进行6样本分析,按“血浆样品预处理”项下操作,测得样品浓度,计算相对误差(RE%)。结果表明未处理的血浆样品室温放置4h稳定,血浆样品处理后进样室(15℃)放置6h稳定,血浆样品经过3次冷冻(-80℃)-解冻循环后稳定,血浆样品长期冷冻(-80℃)放置10d稳定。
实施例2羟基酪醇在Beagle犬体内药代动力学实验
试验采用两周期交叉试验设计。4只Beagle犬,雄性,随机分成2组。禁食12h后,静注组静脉注射给予羟基酪醇溶液,口服组口服给予羟基酪醇胶囊,给药剂量14mg/kg。各组于给药后0.083、0.167、0.333、0.5、1、2、4、6、8和24h自犬前肢静脉取血约1.5mL。
血液样本采集后置于冰上,并于1h之内离心分离血浆(离心条件:8000rpm,10min,2-8℃)。14天后,两组动物交叉给药,按上述方法取静脉血并制备血浆。收集的血浆分析前存放于-80℃冰箱内。
测定血浆生物样品中羟基酪醇采用高效液相色谱-串联质谱(LC-MS/MS)的高通量测定方法。具体为:
1、实验材料和仪器
羟基酪醇(批号:20181207,含量:97.19%),山东省药学科学院生物药物研究所提供;甲苯磺丁脲(批号:#H1401054,含量:≧99%),上海阿拉丁生化科技股份有限公司;乙腈(色谱纯),赛默飞世尔科技有限公司;甲醇(色谱纯),赛默飞世尔科技有限公司;甲酸(色谱纯),天赛默飞世尔科技有限公司;醋酸铵(色谱纯),赛默飞世尔科技有限公司;超纯水,Advantage A10Milli-Q超纯水仪制备,抗坏血酸,东北制药集团股份有限公司。
液相色谱系统:SCIEX Exion型液相色谱系统,美国SCIEX公司;MS/MS系统:SCIEXExion LC-TQ5500三重四级杆串联质谱系统,美国SCIEX公司;AUW-120D型电子分析天平,梅特勒-托利多常州衡器有限公司;LE203E/02型电子分析天平,梅特勒-托利多常州衡器有限公司;Sorvall 21R高速冷冻离心机,美国赛默飞世尔科技公司生产;Vortex-3型漩涡混匀器,德国IKA公司生产;S10型手提式高速分散器,宁波新芝生物科技股份有限公司;Milli-QAdvantageA10超纯水仪,默克化学技术(上海)有限公司。
2、液质检测液相条件:流动相A相为0.1%甲酸+5mM醋酸铵水,B相为乙腈(含0.1%甲酸),梯度洗脱:0~0.7min 2%B,0.7~1.5min 2%→90%B,1.6~2.5min 2%B;色谱柱:Accucore aQ C18(2.1×50mm 2.6μm);流速:0.5mL/min;柱温:40℃;进样室温度:15℃;进样量:1.0μL;样品检测时间:2.5min。
质谱条件:AB SCIEX5500三重四级杆串联质谱系统;离子源:ESI源;负离子模式;气帘气压力:-35psi;碰撞气:-8psi;离子化电压:-5500psi;离子源温度:550℃;喷雾气:-55psi;辅助加热器:-55psi;多重反应监测(MRM)模式;检测离子:m/z 152.9→m/z 123.0(羟基酪醇,CE为-20V,DP为-70V),m/z 269.10→m/z 170.10(甲苯磺丁脲,CE为-50V,DP为-120V)。
血浆样品处理:取100μL血浆样品置于1.5mL离心管中,加入10μL甲苯磺丁脲内标溶液及500μL乙腈溶液,涡旋2min,13000rpm(8℃)离心5min,取上清液1μL进行LC-MS/MS分析。
3、实验结果
Beagle犬分别静注及口服给予羟基酪醇14mg/kg后,每只Beagle犬的血药浓度见表1~2;采用Phoenix WinNonlin8.1药代软件对所测数据进行分析,得到主要的药代动力学参数见表3-4,结果显示,在本试验条件下,以AUC(0-∞)计算,Beagle犬口服羟基酪醇(14mg/kg)后生物利用度为56.90±18.80%。
总体上讲,本发明建立并验证了定量检测Beagle犬血浆样品中羟基酪醇的LC-MS/MS方法,方法的特异性、重现性和准确性均符合药代动力学研究的要求,其中该方法学的定量限为1ng/mL,成功应用于Beagle犬药代动力学研究,进一步推动了羟基酪醇的成药性评价和临床前研究。
表1 Beagle犬分别静脉注射羟基酪醇(14mg/kg)后,血药浓度-时间数据
注:“ND”表示样本浓度低于检测限。
表2 Beagle犬分别口服羟基酪醇(14mg/kg)后,血药浓度-时间数据
注:“NC”表示未获取样本;“ND”表示样本浓度低于检测限;“NA”表示无法获取数据。
表3 Beagle犬静脉注射羟基酪醇(14mg/kg)后,羟基酪醇主要药动学参数
表4 Beagle犬口服羟基酪醇(14mg/kg)后,羟基酪醇主要药动学参数
Claims (6)
1.一种测定Beagle犬血浆中羟基酪醇的方法,其特征在于,具体包括如下步骤:
(1)血浆样品预处理:取血浆样品加入甲苯磺丁脲内标溶液,再加入乙腈溶液沉淀蛋白,涡旋离心后取上清液;
(2)采用高效液相色谱法-电喷雾离子化串联质谱(LC-MS/MS)法检测羟基酪醇在Beagle犬血浆中的浓度:所用的液相系统条件为:流动相为0.1%甲酸+5mM醋酸铵水和含0.1%甲酸的乙腈,梯度洗脱;质谱系统采用ESI源,负离子扫描模式;
该方法可对Beagle犬血浆样品中羟基酪醇进行定量检测,定量检测范围为1-2000ng/mL。
2.如权利要求1所述的一种测定Beagle犬血浆中羟基酪醇的方法,其特征在于步骤(1)具体为:取100μL血浆样品置于1.5mL离心管中,加入10μL甲苯磺丁脲内标溶液及500μL乙腈溶液,涡旋2min,13000rpm(8℃)离心5min,取上清液作为待测液。
3.如权利要求1所述的一种测定Beagle犬血浆中羟基酪醇的方法,其特征在于,LC-MS/MS法中液相系统条件为流动相:A相为0.1%甲酸+5mM醋酸铵水,B相为乙腈(含0.1%甲酸),梯度洗脱:0~0.7min2%B,0.7~1.5min2%→90%B,1.6~2.5min2%B。
4.如权利要求1所述的一种测定Beagle犬血浆中羟基酪醇的方法,其特征在于,LC-MS/MS法中液相系统条件为色谱柱为AccucoreaQC18色谱柱(2.1×50mm,2.6μm);流速:0.5mL/min;柱温:40℃;进样室温度:15℃;进样量:1.0μL;进样时间:2.5min。
5.如权利要求1所述的一种测定Beagle犬血浆中羟基酪醇的方法,其特征在于,LC-MS/MS法中质谱系统条件为:ABSCIEX5500三重四级杆串联质谱系统;离子源:ESI源;负离子模式;气帘气压力:-35psi;碰撞气:-8psi;离子化电压:-5500psi;离子源温度:550℃;喷雾气:-55psi;辅助加热气:-55psi;多重反应监测(MRM)模式。
6.如权利要求1所述的一种测定Beagle犬血浆中羟基酪醇的方法,其特征在于,羟基酪醇的m/z母离子→子离子为152.9→123.0,CE(碰撞能量)为-20V,DP(去簇电压)为-70V;甲苯磺丁脲的m/z母离子→子离子为269.1→170.1,CE为-50V,DP为-120V。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010321750.4A CN112198269A (zh) | 2020-04-22 | 2020-04-22 | 一种测定Beagle犬血浆中羟基酪醇的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010321750.4A CN112198269A (zh) | 2020-04-22 | 2020-04-22 | 一种测定Beagle犬血浆中羟基酪醇的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112198269A true CN112198269A (zh) | 2021-01-08 |
Family
ID=74004908
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010321750.4A Pending CN112198269A (zh) | 2020-04-22 | 2020-04-22 | 一种测定Beagle犬血浆中羟基酪醇的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112198269A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115078568A (zh) * | 2022-05-23 | 2022-09-20 | 山东省药学科学院 | 一种测定羟基酪醇含量及纯度的超高效液相色谱方法 |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1582512A1 (en) * | 2004-03-31 | 2005-10-05 | Cognis IP Management GmbH | Process for obtaining hydroxytyrosol from olive leaves extracts |
US20160184434A1 (en) * | 2013-08-09 | 2016-06-30 | Suntory Holdings Limited | Agent for promoting in vivo absorption of hydroxytyrosol and derivatives thereof and use of same |
CN107019672A (zh) * | 2017-03-28 | 2017-08-08 | 中国林业科学研究院林产化学工业研究所 | 一种富含羟基酪醇和毛蕊花苷油橄榄多酚提取物长循环脂质体的制备方法 |
CN107158025A (zh) * | 2017-05-05 | 2017-09-15 | 山东省药学科学院 | 一种含有羟基酪醇的降血脂组合物及其应用 |
CN107703221A (zh) * | 2017-08-21 | 2018-02-16 | 山东省药学科学院 | 一种同时测定Beagle犬血浆中雷贝拉唑钠及其代谢物的方法 |
CN109942429A (zh) * | 2019-03-01 | 2019-06-28 | 陕西科技大学 | 新型羟基酪醇莽草酸酯化合物及其在制备心脑血管药物中的应用 |
CN110133169A (zh) * | 2019-04-16 | 2019-08-16 | 天津力生制药股份有限公司 | 一种采用液质联用检测人血浆中呋塞米的方法及应用 |
EP3590356A1 (en) * | 2018-07-05 | 2020-01-08 | Uriach Consumer Healthcare, S.L. | Composition for controlling and reducing blood cholesterol levels |
-
2020
- 2020-04-22 CN CN202010321750.4A patent/CN112198269A/zh active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1582512A1 (en) * | 2004-03-31 | 2005-10-05 | Cognis IP Management GmbH | Process for obtaining hydroxytyrosol from olive leaves extracts |
US20160184434A1 (en) * | 2013-08-09 | 2016-06-30 | Suntory Holdings Limited | Agent for promoting in vivo absorption of hydroxytyrosol and derivatives thereof and use of same |
CN107019672A (zh) * | 2017-03-28 | 2017-08-08 | 中国林业科学研究院林产化学工业研究所 | 一种富含羟基酪醇和毛蕊花苷油橄榄多酚提取物长循环脂质体的制备方法 |
CN107158025A (zh) * | 2017-05-05 | 2017-09-15 | 山东省药学科学院 | 一种含有羟基酪醇的降血脂组合物及其应用 |
CN107703221A (zh) * | 2017-08-21 | 2018-02-16 | 山东省药学科学院 | 一种同时测定Beagle犬血浆中雷贝拉唑钠及其代谢物的方法 |
EP3590356A1 (en) * | 2018-07-05 | 2020-01-08 | Uriach Consumer Healthcare, S.L. | Composition for controlling and reducing blood cholesterol levels |
CN109942429A (zh) * | 2019-03-01 | 2019-06-28 | 陕西科技大学 | 新型羟基酪醇莽草酸酯化合物及其在制备心脑血管药物中的应用 |
CN110133169A (zh) * | 2019-04-16 | 2019-08-16 | 天津力生制药股份有限公司 | 一种采用液质联用检测人血浆中呋塞米的方法及应用 |
Non-Patent Citations (8)
Title |
---|
P. DEL BOCCIO ET AL.: "Liquid chromatography–tandem mass spectrometry analysis of oleuropein and its metabolite hydroxytyrosol in rat plasma and urine after oral administration", 《JOURNAL OF CHROMATOGRAPHY B》 * |
S.-T. WANG, ET AL.: "Efficient extraction and sensitive LC-MS quantification of hydroxytyrosol in wine, oil and plasma", 《FOOD CHEMISTRY》 * |
ZHANG WEI, ET AL.: "Pharmacokinetics of acteoside following single dose intragastric and intravenous administrations in dogs", 《CHINESE JOURNAL OF NATURAL MEDICINES》 * |
何文娟等: "CYP2D6 抑制剂对艾瑞昔布大鼠体内药代动力学的影响", 《中国药业》 * |
徐保鑫等: "女贞子中橄榄苦苷与羟基酪醇在大鼠血浆中的药动学差异", 《中成药》 * |
李云飞等: "红景天苷衍生物对抗抑郁及促学习记忆作用的影响", 《世界临床药物》 * |
王子璐等: "芦荟大黄素的体外肝微粒体代谢动力学和代谢酶表型研究", 《国际药学研究杂志》 * |
邢世华: "基于胃肠道代谢的黄芩、连翘药效物质基础研究", 《中国优秀博硕士学位论文全文数据库(博士)医药卫生科技辑》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115078568A (zh) * | 2022-05-23 | 2022-09-20 | 山东省药学科学院 | 一种测定羟基酪醇含量及纯度的超高效液相色谱方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Hua et al. | Determination of berberine in human plasma by liquid chromatography–electrospray ionization–mass spectrometry | |
Zhang et al. | LC–MS/MS determination and pharmacokinetic study of seven flavonoids in rat plasma after oral administration of Cirsium japonicum DC. extract | |
Chang et al. | Simultaneous determination and pharmacokinetic study of six flavonoids from Fructus Sophorae extract in rat plasma by LC–MS/MS | |
CN106990185B (zh) | 一种同时测定血浆中六种酪氨酸激酶抑制剂浓度的方法 | |
CN109239224B (zh) | 酸枣仁水提物中9种入血成分的同时定量测定方法 | |
Ma et al. | Simultaneous determination of tetrahydropalmatine, protopine, and palmatine in rat plasma by LC-ESI-MS and its application to a pharmacokinetic study | |
CN108828077B (zh) | 一种同时检测血浆中卡培他滨及其代谢产物的试剂盒及其检测方法与应用 | |
CN111562322B (zh) | 一种血样中五种抗肿瘤药物的富集检测方法及应用 | |
Sheng et al. | Pharmacokinetic and excretion study of three secoiridoid glycosides and three flavonoid glycosides in rat by LC–MS/MS after oral administration of the Swertia pseudochinensis extract | |
US20230138381A1 (en) | A lc-ms/ms method for measurement of aloesin in rat plasma | |
Liu et al. | Development and validation of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of raddeanin A in rat plasma and its application to a pharmacokinetic study | |
CN110031568B (zh) | 一种测定人血浆中沙库巴曲、去乙基沙库巴曲和缬沙坦浓度的方法 | |
CN112198269A (zh) | 一种测定Beagle犬血浆中羟基酪醇的方法 | |
Li et al. | Development and validation of a LC-ESI–MS/MS method for the determination of swertiamarin in rat plasma and its application in pharmacokinetics | |
Yang et al. | High-performance liquid chromatography–electrospray ionization mass spectrometry determination of sodium ferulate in human plasma | |
Chen et al. | Determination and pharmacokinetics of DT-13 in rat plasma by LC–MS | |
Sun et al. | Simultaneous determination of acetylpuerarin and puerarin in rat plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study following intravenous and oral administration | |
Wen et al. | A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of hydroxysafflor yellow A in human plasma: Application to a pharmacokinetic study | |
Xia et al. | Development and validation of a sensitive liquid chromatography–tandem mass spectrometry method for the determination of paeoniflorin in rat brain and its application to pharmacokinetic study | |
Yin et al. | Simultaneous determination of six alkaloid components in rat plasma and its application to pharmacokinetic study of Danmu preparations by an ultra fast liquid chromatography–electrospray ionization-tandem mass spectrometry | |
CN109298081B (zh) | 一种新利司他中杂质a生物样品的测定方法 | |
CN112540138A (zh) | 一种血浆中丹酚酸b、阿司匹林、水杨酸的联合定量测定方法 | |
CN105806985B (zh) | 一种马钱子苷元生物样品的测定方法 | |
Sun et al. | Determination of hydromorphone in human plasma by a sensitive RP-HPLC–ESI-MS method and its application to a clinical pharmacokinetic study in postoperative patients after low dose intravenous administration with infusion pump | |
Yu et al. | Determination of oroxin A, oroxin B, oroxylin A, oroxyloside, chrysin, chrysin 7-O-beta-gentiobioside, and guaijaverin in mouse blood by UPLC-MS/MS and its application to pharmacokinetics |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210108 |