CN109096316B - 氘代-3-硝基苯硼酸及其制备方法与应用 - Google Patents
氘代-3-硝基苯硼酸及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了氘代‑3‑硝基苯硼酸及其制备方法与应用。该化合物与单甘油酯进行衍生化反应后，可增加单甘油酯的质谱响应。同时，利用硼原子特有的同位素分布特征，提高了单甘油酯检测的特异性和鉴定的准确性。通过与轻标试剂联合使用，可用于单甘油酯在复杂生物样本中的检测和相对定量分析。
Description
技术领域
本发明属于分析检测领域，具体涉及一种氘代-3-硝基苯硼酸及其制备方法与应用。
背景技术
单甘油酯(MAG)是一类参与体内多种生理过程的活性物质。研究表明，单甘油酯与多种疾病有密切的关系，例如糖尿病、肥胖症、癌症等。目前多采用间接检测的方法对复杂生物样品中单甘油酯混合物进行鉴定，即将单甘油酯降解为甘油和对应的游离脂肪酸，通过薄层色谱将游离脂肪酸分离后，用气相色谱-质谱联用的方法对所得游离脂肪酸混合物进行分离及鉴定。这种方法耗时且前处理过程复杂，易造成样品损失。同时，对单甘油酯整体分子的质谱检测，受单甘油酯结构中羟基基团电离性质的限制，仅能在负离子模式下进行检测，降低了检测的灵敏度，同时在二级质谱中易丢失结构信息。除此之外，单甘油酯的定量受到无合适内标或氘代单甘油酯不易获得等因素限制，增加了其在复杂生物样品中检测、鉴定及定量的难度。
发明内容
本发明的目的是针对现有技术存在的不足，提供氘代-3-硝基苯硼酸，其为针对单甘油酯羟基基团反应的衍生化试剂，该化合物与单甘油酯进行衍生化反应后，由于硼元素的吸电子特性，衍生化后所得产物带负电荷，可增加衍生化产物在负离子模式下的质谱响应；同时，利用硼特有的同位素分布特征，提高了单甘油酯检测的特异性和鉴定的准确性。本发明的第二目的是提供氘代-3-硝基苯硼酸的制备方法。本发明的第三目的是提供氘代-3-硝基苯硼酸在复杂生物样品中的应用。
为实现上述目的，本发明采用以下技术方案：
氘代-3-硝基苯硼酸，其结构式如式I所示：
第二方面，提供上述氘代-3-硝基苯硼酸的制备方法，包括以下步骤；
(1)氘代-3-硝基苯胺溶于水，得到浓度为0.36mmol/mL的溶液，加入5％盐酸溶液，两者体积比为1：0.6，室温搅拌1分钟得到混合溶液；
(2)将亚硝酸钠溶于水后，得到浓度为0.86mmol/mL的溶液，逐滴加入上述步骤(1)的混合溶液中，混合溶液与亚硝酸钠溶液的体积比为3.2：1，0℃搅拌15分钟；
(3)向步骤(2)溶液中加入B2(OH)4、醋酸钠和步骤(2)溶液3倍体积的水，使得B2(OH)4的浓度为0.09mmol/mL，醋酸钠的浓度为0.09mmol/mL，室温搅拌20分钟；
(4)向步骤(3)溶液中加入3倍体积的乙酸乙酯，用饱和碳酸钾调节pH 值为8；混合物分层后，有机层用饱和碳酸钾萃取4次，合并水相用5％盐酸溶液调节pH值至6，用乙酸乙酯萃取3次，合并有机相，用水洗涤2次，再用饱和食盐水洗涤，加入无水硫酸钠干燥，抽滤，减压蒸去溶剂得到灰白色固体氘代-3-硝基-苯硼酸。
其反应机理如下所示：
第三方面，提供上述氘代-3-硝基苯硼酸作为一种衍生化试剂在单甘油酯超高效液相色谱-质谱检测中的应用，所述氘代-3-硝基苯硼酸对单甘油酯的衍生化反应为3-硝基苯硼酸在溶液中与氢氧根结合形成负离子形式后，与单甘油酯中的二羟基基团结合，脱去两分子H2O，形成一个稳定的五元环，其反应路线如下所示：
其中，R1为脂肪酸的碳链部分。
优选地，所述应用是上述氘代-3-硝基苯硼酸(d4-NPB)与3-硝基-苯硼酸 (d0-NPB)等量混合后联合使用，将样品中单甘油酯衍生后，通过超高效液相色谱-质谱在样品中检测单甘油酯。
优选地，所述应用是上述氘代-3-硝基苯硼酸(d4-NPB)与3-硝基-苯硼酸 (d0-NPB)等量分别标记不同生理状态下的样本，通过衍生化产物的质谱响应比值，实现单甘油酯在不同样本中的相对定量分析。
d0-NPB和d4-NPB的化学结构式及衍生化反应机理如下所示：
R1为脂肪酸的碳链部分。
衍生化反应原理：
3-硝基苯硼酸在溶液中与氢氧根结合形成负离子形式后，与单甘油酯中的二羟基基团结合，脱去两分子H2O，形成一个稳定的五元环，成功固定了单甘油酯分子的原有结构，使其不易转变为其异构体。衍生化产物以负离子的形式，且具有硼原子特有的同位素分布特征，更容易在负离子模式下被检测，提高了质谱的响应。
本发明具有以下优点：
1、在本发明中，合成了针对单甘油酯羟基基团反应的衍生化试剂，氘代- 3-硝基苯硼酸(d4-NPB)。由于硼元素的吸电子特性，衍生化后所得产物带负电荷，增加了其在负离子模式下的质谱响应。同时，利用硼原子特有的同位素分布特征，提高了单甘油酯检测的特异性和鉴定的准确性。
2、氘代-3-硝基苯硼酸(d4-NPB)与轻标(即d0-NPB)衍生化试剂等比例混合后进行衍生化反应，使衍生化产物在质谱图中展现质量数差值为4Da的双峰特征，能够快速准确地在复杂样品中识别单甘油酯。
3、将轻标及重标衍生化试剂分别用于不同生理状态下的样品中的单甘油酯衍生化，通过对轻/重标产物的质谱响应比值的测定，可实现不同生理状态下单甘油酯的相对定量分析。
4、本发明使用衍生化方法检测单甘油酯，操作更加简单且质谱检测灵敏度更高。
附图说明
图1为氘代-3-硝基苯硼酸(d4-NPB)与3-硝基-苯硼酸(d0-NPB)等量混合后联合使用检测单甘油酯标准品的质谱图。
图2为氘代-3-硝基苯硼酸与3-硝基-苯硼酸等量分别标记不同样本检测所含单甘油酯的质谱图。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。
【实施例1】氘代-3-硝基-苯硼酸的制备
氘代-3-硝基苯胺(50mg，0.36mmol)溶于水(1mL)，加入5％盐酸溶液 (0.6mL，0.90mmol)，室温搅拌1分钟得到混合溶液。将亚硝酸钠(30mg， 0.43mmol)溶于水(0.5mL)后，逐滴加入上述混合溶液中，0℃搅拌15分钟，加入B2(OH)4(64.9mg，0.72mmol)，醋酸钠(59.36mg，0.72mmol)和水(6mL)，室温搅拌20分钟，加入乙酸乙酯(25mL)，用饱和碳酸钾调节 pH值为8。混合物分层后，有机层用饱和碳酸钾(10mL×4)萃取，合并水相用5％盐酸溶液调节pH值至6，用乙酸乙酯(10mL×3)萃取，合并有机相，用水(10mL×2)洗涤，再用饱和食盐水(10mL)洗涤，加入无水硫酸钠干燥，抽滤，减压蒸去溶剂得到灰白色固体氘代-3-硝基-苯硼酸(18mg，产率 29％)。1H NMR(300MHz,DMSO-d6)δ(ppm):8.49(s,2H).13C NMR(75MHz,DMSO-d6)δ(ppm):147.88,140.65,131.94,129.10,124.50.HRMS:C6H3D4BNO5 [M-H]+,calcd170.0568found 170.0625.m.p.:283-285℃。
【实施例2】单甘油酯标准品衍生化及超高效液相色谱-质谱(UPLC-MS)检测
1.精密称取棕榈酸单甘油酯(PG)、油酸单甘油酯(OG)、花生四烯酸单甘油酯(AG)、亚油酸单甘油酯(LG)、硬脂酸单甘油酯(SG)5种标准品，用甲醇溶解，制备成浓度为100μM的混标溶液。
2.精密称取d0-NPB及d4-NPB固体，用甲醇溶解后，制备成10mM的母液。
3.取100μL混标溶液，加入制备好的10mM的d0-NPB溶液和d4-NPB溶液各10μL进行衍生化反应，反应条件为37℃，400rpm，反应2小时。
4.衍生化反应结束后，于4℃，18,000rpm离心10分钟，取上清液50μL进行超高效液相色谱-质谱分析。
进样分析后的结果如图1所示，由液相分离结果可以看出，单甘油酯经过 d4-NPB衍生化后并不会影响其液相色谱行为，与d0-NPB标记的衍生化产物同时被洗脱。其次，从质谱中检测到每种单甘油酯都有质量数差值为4Da的双峰，同时具有B元素的特异性同位素分布，这有利于快速鉴定复杂样本中的单甘油酯。采用d0/d4-NPB 1:1比例混合后衍生化，所得的产物质谱信号波动在 10％以内，证明所合成的d4-NPB衍生化效率与d0-NPB效率无显著差别。
【实施例3】肝脏组织衍生化后进样来验证氘代试剂的作用
肥胖小鼠肝脏作为实验组使用d4-NPB衍生化，正常小鼠肝脏作为对照组使用d0-NPB衍生化，来验证氘代试剂的作用。
具体过程如下：
1.称取肥胖小鼠肝脏和正常小鼠肝脏各60mg，分别加入400μL甲醇，4000 rpm，匀浆1分钟，振荡10分钟，-20℃静置20分钟后，于4℃，18,000 rpm离心10分钟取出上清液备用。
2.精密称取d0-NPB及d4-NPB固体，用甲醇溶解后，制备成10mM的母液。
3.将肥胖组上清液取100μL加入10μL制备好的d4-NPB溶液进行衍生化，正常组上清液取100μL加入10μL制备好的d0-NPB溶液进行衍生化，衍生化条件为37℃，400rpm，反应2小时。
4.衍生化结束后，于4℃，18,000rpm离心10分钟，各取上清液50μL 进行超高效液相色谱-质谱分析。液相色谱串联质谱联用系统：超高效液相色谱仪(Waters ACQUITY UPLCI-Class),四级杆飞行时间质谱仪(Waters Synapt G2Si Q-TOF)。
进样分析后的结果如图2所示，检测到五种单甘油酯，分别为、PG、 OG、SG、肉豆蔻酸单甘油酯(Monomyristin)及月桂酸甘油酯(GML)。其中， PG、欧OG、SG三种单甘油酯与肥胖症之间存在密切的关系，并且在脂质代谢途径中有很重要的作用，所以选取了PG、OG、SG三种物质来进行分析。
首先由液相分离结果看出，PG、OG、SG的d0-NPB衍生化产物的保留时间分别为4.33、4.51、4.43分钟，与d4-NPB衍生化产物的保留时间相同。其次从质谱图中，我们可以发现PG主要以水分子加合物H2O的形式存在，检测到被d0/d4-NPB标记的PG衍生化产物的m/z分别为496.3和500.3。OG的 d0/d4-NPB标记的衍生化产物检测到两种形式，产物m/z分别为504.3/508.3 (d0/d4)，水分子加合物m/z分别为522.3/526.3(d0/d4)。而SG m/z分别为506.3/510.3(d0/d4)。从结果发现，每种单甘油酯的质谱图中依旧展示了质量数差值为4Da的双峰，具有B元素的特异性同位素分布。从质谱图中大致可以看出，肥胖小鼠肝脏和正常小鼠肝脏中的PG含量基本相同，肥胖小鼠肝脏中的 OG含量比正常小鼠肝脏高，肥胖小鼠肝脏中的SG含量比正常小鼠肝脏低。由此，使用d0/d4-NPB试剂衍生化可用于分析不同生物样本中单甘油酯的相对含量。
Claims (1)
1.氘代-3-硝基苯硼酸作为一种衍生化试剂在单甘油酯超高效液相色谱-质谱检测中的应用，所述氘代-3-硝基苯硼酸对单甘油酯的衍生化反应为3-硝基苯硼酸在溶液中与氢氧根结合形成负离子形式后，与单甘油酯中的二羟基基团结合，脱去两分子H2O，形成一个稳定的五元环，其反应路线如下所示：
其中，R1为脂肪酸的碳链部分，其特征在于：
所述应用是所述的氘代-3-硝基苯硼酸(d4-NPB)与3-硝基-苯硼酸(d0-NPB)等量混合后联合使用，将样品中单甘油酯衍生，通过超高效液相色谱-质谱检测样品中单甘油酯；
或者所述应用是所述的氘代-3-硝基苯硼酸与3-硝基-苯硼酸等量分别标记不同样本，通过衍生化产物的质谱响应比值，实现单甘油酯在不同样本中的相对定量分析。
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