CN115389678A - 色胺类新精神活性物质4-ho-dipt体内生物标记物及其应用 - Google Patents
色胺类新精神活性物质4-ho-dipt体内生物标记物及其应用 Download PDFInfo
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Abstract
本发明公开色胺类新精神活性物质4‑HO‑DIPT体内生物标记物及其应用,应用高效液相串联静电轨道离子阱高分辨质谱检测结合斑马鱼模型,通过筛查4‑HO‑DIPT的体内代谢产物,分析4‑HO‑DIPT的生物标记物。本发明克服色胺类新精神活性物质4‑HO‑DIPT在体内代谢速度快,在生物检材中的母体药物含量一般较低,提供一种利用体内代谢标记物进行检测,精确度高,灵敏度高,结果准确可靠。本发明所需的检测样本生物量较少,仅为100μl。
Description
技术领域
本发明涉及色胺类新精神活性物质4-HO-DIPT体内生物标记物及其应用,特别应用高效液相串联静电轨道离子阱高分辨质谱检测结合斑马鱼模型,通过筛查 4-HO-DIPT的体内代谢产物,分析4-HO-DIPT的生物标记物。
背景技术
色胺类新精神活性物质是一类含有色胺结构框架、具有致幻作用的化合物。许多古老的色胺类化合物以其强烈的致幻作用而闻名,如来自致幻蘑菇的赛洛西宾(Psilocybin)、赛洛新(Psilocin)和来自死藤水的二甲基色胺(DMT)。以赛洛新和赛洛西宾为代表的致幻蘑菇是传统的、天然的一类神经致幻剂,服用后能使人产生致幻作用,产生时间和空间的变形和幻觉。近些年,新型色胺类物质大量合成,它们具有与传统管制药物相似的化学结构和性质,却披着“合法的”外衣,已经成为不法分子的新目标。色胺类物质(tryptamines)具有吲哚环结构,是由吡咯环和苯环组成的融合双环,通过二碳侧链与氨基相连,目前已经确认的色胺类物质有45种。
1999年,自N,N-二异丙基-5-甲氧色胺(N,N-Diisopropyl-5-methoxy-tryptamine,5-MeO-DiPT),俗称‘Foxy’滥用后,新型色胺类致幻剂不断出现,经互联网销售而流行。根据UNODC的报告,直到2012年,约10%的新精神活性物质为色胺类,仅次于合成大麻类(23%)、苯乙胺类(23%)和合成卡西酮类(18%)。目前对于色胺类新精神活性物质分析方法的研究较少,仅有一些文献对单个色胺类化合物进行分析,如:BRANDT等对DALT的研究、MATTIN等对赛洛新的分析、 FABREGAT等对5-MeO-DiPT和5-MeO-MiPT的体内代谢研究,有少量文献报道对多种色胺类化合物进行分析,如MEYET等对37种色胺类衍生物的定性定量分析等。
因此对于色胺类新精神活性物质及其代谢物的检测刻不容缓。色胺类新精神活性物质进入人体后代谢较快,生物检材中其母药可检测的含量较低,因此确定色胺类新精神活性物质的生物代谢标记物尤为重要。目前国内外对于色胺类新精神活性物质的代谢研究包括体内和体外代谢。体外代谢方法主要包括人肝细胞培养和肝微粒体孵育实验,体内代谢则以大鼠模型和斑马鱼模型为主。
4-HO-DIPT作为一种通过人工合成的色胺类新精神活性物质,目前暂未列入新精神活性物质管控名单。然而,目前对4-HO-DIPT的代谢产物尚不清楚,在人体在尿液中,4-HO-DIPT的主要代谢物仅鉴定出有4-HO-DIPT氧化物和4-HO-DIPT 的吡啶-N-葡萄糖苷酸。因此,通过合适的手段对4-HO-DIPT的代谢途径进行鉴定分析对于研究4-HO-DIPT的毒性机理具有重大意义。
发明内容
本发明的目的是针对现有技术的不足,提供色胺类新精神活性物质 4-HO-DIPT生物标记物。
表1:检测色胺类新精神活性物质4-HO-DIPT的体内生物标记物
本发明的第二个目的是提供上述利用上述体内生物标记物在非诊断或治疗目的的检测色胺类新精神活性物质4-HO-DIPT上的应用。
本发明的第三个目的是提供上述利用上述体内生物标记物的一种非诊断或治疗目的的色胺类新精神活性物质4-HO-DIPT检测方法,具体是:
(1)取100μl尿液加入3倍体积的乙腈,涡旋,时间不少于30s,大于 12000rpm的转速离心一定时间,取上清液;
(2)将上述滤液进行高效液相串联静电轨道离子阱高分辨质谱检测,若同时检测到表1三种标记物则认为事先服用色胺类新精神活性物质4-HO-DIPT。
所述液相色谱条件:
色谱柱:WatersCORTECSUPLCT3(2.1×150mm,1.6μm);
柱温:30℃;
流动相:流动相A为体积含量为0.1%的甲酸水,流动相B为体积含量0.1%的甲酸乙腈;
流动相洗脱梯度:0-1min:流动相A和流动相B体积比保持99:1;1-8min:流动相A和流动相B体积比由99:1匀速渐变至1:99;8-10min:流动相A和流动相B体积比保持1:99;10-12min:流动相A和流动相B体积比保持99:1;
流速为0.3mL/min,进样量为5μL;
质谱条件:电喷雾离子源(ESI),正离子采集模式。离子源参数:碰撞气:氮气;喷雾电压:3kV;雾化温度:320℃;离子传输管温度:350℃;鞘气流量: 40arb;辅助气流量:10arb。
采用FullMS-ddMS2扫描模式采集数据,全扫描数据采集参数:分辨率: 35000;自动增益控制(AGC):1e6;最大注射时间(MIT):100ms;扫描范围:70to 1000m/z。二级质谱数据采集参数:分辨率:17500;AGC:2e5;MIT:60ms;归一化碰撞能量(NCE):17.5,35,52.5。
本发明的第四个目的是提供一种色胺类新精神活性物质4-HO-DIPT检测的工具,包括上述标记物。
作为优选,所述工具指代独立试剂或试剂盒。
本发明具有如下优点:
1)本发明克服色胺类新精神活性物质4-HO-DIPT在体内代谢速度快,在生物检材中的母体药物含量一般较低,提供一种利用体内代谢标记物进行检测,精确度高,灵敏度高,结果准确可靠。
2)本发明所需的检测样本生物量较少,仅为100μl。
附图说明
图1是4-HO-DIPT的色谱图,其中(a)标准溶液,(b)注射完成15min后斑马鱼体内的样品,(c)空白斑马鱼样品;
图2是4-HO-DIPT的二级质谱图;
图3是斑马鱼体内代谢产物二级质谱图及推导碎裂模式;
图4是斑马鱼体内代谢途径示意图。
具体实施方式
下面结合具体实施例和附图对本发明做进一步地分析。
实施例1:高效液相串联静电轨道离子阱高分辨质谱检测结合斑马鱼模型,得到4-HO-DIPT的体内代谢产物:
(1)准备12条六个月大的成年斑马鱼,随机分为空白对照组和实验组,每组6条,斑马鱼在实验前进行24h的禁食处理;
(2)使用25μL规格的微量注射器对成年斑马鱼进行腹腔注射,实验组注射10μL浓度为50μg/ml的4-HO-DIPT溶液(溶剂为超纯水),空白对照组则注射10μL超纯水;
(3)注射完成后分别将实验组和空白对照组斑马鱼放入养殖系统水中, 15min后取出放入冰块中处死,用超纯水清洗3次后放入2ml规格研磨管于-80℃冰箱中保存。
(4)在研磨管中加入适量研磨珠(3.2mm)及乙腈甲醇(2:1)溶液300μL,在冷冻研磨仪中进行处理。研磨机参数设置:频率65Hz,温度-50℃,运行时间30s,中断时间30s,循环10次。然后将样品以13500rpm离心5min后取上清液,通过0.22μm有机相针式滤膜过滤后进样。
得到4-HO-DIPT的8种代谢产物(见表2)
色谱方法:选择CORTECSUPLCT3(2.1×150mm,1.6μm;美国Waters公司)。流动相A:0.1%甲酸水溶液;流动相B:0.1%甲酸乙腈溶液。梯度洗脱程序:0-1min: 1%B;1-8min:1%-99%B;8-10min:保持99%B;10-12min:1%B。流速为0.3ml/min,进样量为5μL,柱温30℃,自动进样盘10℃。
质谱方法:电喷雾离子源(ESI),正离子采集模式。离子源参数:碰撞气:氮气;喷雾电压:3kV;雾化温度:320℃;离子传输管温度:350℃;鞘气流量: 40arb;辅助气流量:10arb。
采用FullMS-ddMS2扫描模式采集数据,全扫描数据采集参数:分辨率:35000;自动增益控制(AGC):1e6;最大注射时间(MIT):100ms;扫描范围:70to1000 m/z。二级质谱数据采集参数:分辨率:17500;AGC:2e5;MIT:60ms;归一化碰撞能量(NCE):17.5,35,52.5。
实施例2:分析4-HO-DIPT体外代谢物
在总离子流图中输入母药精确质量数,提取色谱峰;对比标准品、未加标准目标物和经腹腔注射15min后样品的色谱峰响应值,见图1,其中(a)标准溶液, (b)注射完成15min后斑马鱼体内的样品,(c)空白斑马鱼样品。
图2是4-HO-DIPT的二级质谱图;
图3是斑马鱼体内代谢产物二级质谱图及推导碎裂模式;
图4是斑马鱼体内代谢途径示意图。
4-HO-DIPT的分子式为C16H24N2O,质荷比为m/z261.19614;二级质谱图主要碎片离子为m/z160.07556、m/z114.12791、m/z72.08141。
M1(C16H24N2O2)和M2(C16H24N2O3)为4-HO-DIPT发生羟基化的Ⅰ相代谢产物,其中M1保留时间为4.97min,质子化的分子离子[M+H]+为m/z277.19073,与母药相比多了15.99503Da,碎片离子为m/z176.07048、114.12790、72.08140,与母药的碎片离子进行对比,可以确定羟基化反应发生在吲哚结构上。
M2则为4-HO-DIPT发生了二羟基化后的代谢产物,保留时间为4.58min,质子化的分子离子[M+H]+为m/z293.18542,比4-HO-DIPT增加了31.98968Da,再结合其碎片离子m/z192.06532、114.12785、72.08141,可以确定发生二羟基化的位点在吲哚结构上。
M3(C16H26N2O2)、M4(C16H26N2O3)是4-HO-DIPT发生了水合反应的Ⅰ相代谢产物,其中M3的保留时间为4.80min,其m/z为279.20657,比4-HO-DIPT增加了 18.01083Da,其特征碎片离子m/z分别是178.08615、160.07555、114.12790、 72.06141,可以推测出水合反应在吲哚结构上发生。
M4是吲哚环上发生了羟基化反应后再发生水合反应。其保留时间为4.44min, m/z为295.20120,对比M1增加了18.01047Da,形成的特征碎片离子 m/z194.08147、176.07056、114.12788、72.06141,所以M4是由M1进一步在吲哚环上发生水合反应形成的。
表2 4-HO-DIPT及代谢产物分析
M5(C16H22N2O3)为4-HO-DIPT形成了羧酸的Ⅰ相代谢产物,其保留时间为 5.07min,质子化的分子离子[M+H]+为m/z291.16986,比4-HO-DIPT增加了 29.97412Da,可以推测出是增加两个氧并脱去两个氢所得,其特征碎片离子m/z 为190.04970、114.12789、72.08141,与4-HO-DIPT对比可以确定羧酸形成的位置是在吲哚环结构。
M6(C22H32N2O7)和M7(C22H32N2O8)是4-HO-DIPT与葡萄糖醛酸结合形成的Ⅱ相代谢产物。M6的保留时间是4.82min,其m/z437.22769,比4-HO-DIPT增加了 176.03195Da,碎片离子m/z是261.19577、160.07558、114.12790、72.08142,与4-HO-DIPT完全一致,查阅文献可得,葡萄糖醛酸的结合位点是在吲哚环的羟基上。
M7是在吲哚环上发生了羟基反应之后再结合葡萄糖醛酸,其保留时间为4.66min,其质子化m/z为453.22269,相对于M1增加了176.03196Da,其碎片离子m/z为277.19110、176.07054、114.12796、72.08144,与M1碎片离子一致,可以推测出M7是M1结合葡萄糖醛酸得到的Ⅱ相代谢产物,结合位点在吲哚环的羟基上。
M8是在吲哚环上发生了羟基反应之后再与硫酸结合得到的Ⅱ相代谢产物,其保留时间为5.05min,m/z是357.14722,相对于M1增加了79.95649Da,其碎片离子m/z为277.19064、176.07047、114.12788、72.08140,与M1一致,可以推测出M8是由M1与硫酸结合产生的Ⅱ相代谢产物。
实施例3:排他检测
本发明需同时检测到4-HO-DIPT羟基化、糖苷化、羟基化加糖苷化三种代谢产物,即可确定人体吸食了4-HO-DIPT。由于合成大麻素5-MEO-DIPT经脱甲基化和脱甲基化之后糖苷化可产生与4-HO-DIPT相同分子式的代谢产物。因此将4-HO-DIPT中的一种代谢产物作为其生物标记物,是不能确定人体是否吸食了4-HO-DIPT的。因此如果在生物检材中同时检测到羟基化、糖苷化、羟基化加糖苷化的代谢产物,即可确定人体吸食了4-HO-DIPT。
上述实施例并非是对于本发明的限制,本发明并非仅限于上述实施例,只要符合本发明要求,均属于本发明的保护范围。
Claims (5)
2.如权利要求1所述的色胺类新精神活性物质4-HO-DIPT的体内生物标记物在检测色胺类新精神活性物质4-HO-DIPT上的应用。
3.一种色胺类新精神活性物质4-HO-DIPT检测方法,其特征在于具体是:
(1)取100μl尿液加入3倍体积的乙腈,涡旋,时间不少于30s,大于12000rpm的转速离心一定时间,取上清液;
(2)将上述滤液进行高效液相串联静电轨道离子阱高分辨质谱检测,若同时检测到权利要求1所述的色胺类新精神活性物质4-HO-DIPT的体内生物标记物则认为存在色胺类新精神活性物质4-HO-DIPT,反之则无;
所述高效液相串联静电轨道离子阱高分辨质谱检测参数如下:
液相色谱条件:
色谱柱:Waters CORTECS UPLC T3,尺寸为2.1×150mm,1.6μm;
柱温:30℃;
流动相:流动相A为体积含量为0.1%的甲酸水,流动相B为体积含量0.1%的甲酸乙腈;
流动相洗脱梯度:0-1min:流动相A和流动相B体积比保持99:1;1-8min:流动相A和流动相B体积比由99:1匀速渐变至1:99;8-10min:流动相A和流动相B体积比保持1:99;10-12min:流动相A和流动相B体积比保持99:1;
流速为0.3mL/min,进样量为5μL;
质谱条件:电喷雾离子源ESI,正离子采集模式。离子源参数:碰撞气:氮气;喷雾电压:3kV;雾化温度:320℃;离子传输管温度:350℃;鞘气流量:40arb;辅助气流量:10arb;
采用FullMS-ddMS2扫描模式采集数据,全扫描数据采集参数:分辨率:35000;自动增益控制AGC:1e6;最大注射时间MIT:100ms;扫描范围:70-1000m/z。二级质谱数据采集参数:分辨率:17500;AGC:2e5;MIT:60ms;归一化碰撞能量NCE:17.5,35,52.5。
4.一种色胺类新精神活性物质4-HO-DIPT的检测工具,其特征在于包括权利要求1所述的色胺类新精神活性物质4-HO-DIPT的体内生物标记物。
5.如权利要求4所述一种色胺类新精神活性物质4-HO-DIPT的检测工具,其特征在于所述工具指代独立试剂或试剂盒。
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