CN114509514A - 一种液相色谱-电喷雾电离串联质谱法检测三斑海马中牛磺酸和精氨酸的方法 - Google Patents
一种液相色谱-电喷雾电离串联质谱法检测三斑海马中牛磺酸和精氨酸的方法 Download PDFInfo
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Abstract
本发明公开了一种液相色谱‑电喷雾电离串联质谱法(LC‑ESI‑MS/MS)检测三斑海马中牛磺酸和精氨酸的方法。本发明无须经过HPLC管柱前或后衍生化方法即可侦测牛磺酸和精氨酸的含量，具体分析步骤简单易操作。本发明对海马等海洋天然产物有效成分的研究具有重要意义。
Description
技术领域
本发明属于分析检测技术领域，具体涉及三斑海马中牛磺酸和精氨酸的检测。
背景技术
《本草纲目》记载:“海马，主难产及血气痛；暖水脏，壮阳道；消瘕块，治疗疮肿毒”，故有温肾壮阳，散结消肿之功效；主治阳痿、肾虚作喘、症瘕积聚、瘰疠痰核、跌打损伤，外治痈肿疗疮。近年来的药理研究表明海马不仅有激素样作用，可增强造血功能，还显示了抗癌、抗衰老、抗疲劳和Ca2+阻断等作用。有学者对雄性小鼠连续摄入膨腹海马(H.abdominalis)酶水解物12周后发现，雄性小鼠的精子活力和精子数量增加，睾酮水平升高，主要源于碱性蛋白酶水解产物和胃蛋白酶水解产物(alcalase hydrolysate andpepsin hydrolysate，ALC和PEP)刺激小鼠睾丸间质细胞(睾酮激素主要由此细胞分泌增殖，提高了血清睾酮水平。海马的多样化药学活性，使其成为近年来的研究热点。
牛磺酸(Taurine)和精氨酸(Arginine)是海马中含有的活性成分。检测海马中牛磺酸和精氨酸的含量，有助于对海马进行品种鉴定、质量评价、药效活性检测等。但现有的针对牛磺酸、精氨酸等的检测方法，往往需要经过HPLC管柱前衍生化或柱后衍生化方可实现，操作较为繁琐。
近几年，液相色谱-电喷雾电离串联质谱法(Liquid chromatographyelectrospray ionization tandem mass spectrometry，LC-ESI/MS/MS)广泛地运用于有机小分子化合物例如氨基酸等的检测，其优点为需要的样品的量较少，具有侦测专一性及高敏感度的特点，针对大部分不会被UV吸收的化合物，可利用适当的游离方法使其被质谱仪所检测到。但截至目前并未有利用液相色谱-电喷雾电离串联质谱法检测三斑海马中牛磺酸和精氨酸的报道。
发明内容
本发明的目的在于克服现有技术的不足之处，提供了一种液相色谱-电喷雾电离串联质谱法(LC-ESI-MS/MS)检测三斑海马中牛磺酸和精氨酸的方法，该方法无须经过HPLC管柱前或后衍生化即可检测牛磺酸和精氨酸的含量，从而可以对三斑海马中的牛磺酸、精氨酸成分进行定性、定量检测，以及用于三斑海马的质量鉴定等。
本发明解决其技术问题所采用的技术方案之一是：
一种液相色谱-电喷雾电离串联质谱法检测三斑海马中牛磺酸和精氨酸的方法，包括：
液相条件：Waters CapLC液相色谱系统；色谱柱Waters Atlantis dC18(3μm，75μm×50μm)；进样量：1μL；柱温：25℃；流动相为溶剂A和溶剂B的混合液，所述溶剂A为0.1％九氟戊酸(NFPA)的水溶液，所述溶剂B为0.1％九氟戊酸(NFPA)的乙腈(ACN)溶液；采用梯度洗脱程序：流动相中溶剂B的初始比例为5％，随后在25min内由5％上升至60％，随后在5min内降回5％，接着再平衡5min；流动相流速为6μL/min，待进入色谱柱前流速减至0.3μL/min；
质谱条件：Waters Micromass Q-TOF Ultima Global质谱仪；电离方式：+ESI模式；电压4.50kV，脱溶剂温度290℃；飞行时间检测控制在加速电压9.1kV、锥电压100V、撞击能量10eV；质谱扫描范围为m/z 50～500；选择反应监测模式(SRM)；以子离子的离子层析波峰面积比对标准品，进而计算三斑海马中牛磺酸和精氨酸的含量。
进一步地，采用三斑海马的萃取物进行上述检测；所述萃取方法包括：新鲜三斑海马经冷冻干燥后以酒精萃取，得到酒精萃取物；酒精萃取后剩下的海马以体积比为0.8～1.2:0.8～1.2的二氯甲烷-甲醇混合液连续萃取若干次，得到的萃取液经浓缩后，以水和乙酸乙酯进行液液相分配萃取若干次，分别收集乙酸乙酯层和水层；合并乙酸乙酯层，得到乙酸乙酯萃取物；水层再用正丁醇进行若干次分配萃取，合并正丁醇层，得到正丁醇萃取物；剩余的水层再除去溶剂，得到水萃取物；再以所述酒精萃取物、乙酸乙酯萃取物、正丁醇萃取物和水萃取物进行牛磺酸和精氨酸的检测。
进一步地，牛磺酸的质荷比为126，牛磺酸的子离子的质荷比为108。
优选地，牛磺酸产生子离子所需的最佳撞击能量为27eV。
进一步地，精氨酸的质荷比为175，精氨酸的子离子的质荷比为158。
优选地，精氨酸产生子离子所需的最佳撞击能量为33eV。
一实施例中，牛磺酸和精氨酸的检测限分别为0.313μg/mL和0.174μg/mL。
一实施例中，牛磺酸和精氨酸的定量限分别为0.625μg/mL和0.435μg/mL。
优选地，仪器控制的软件为MassLynx V4.0。
优选地，数据收集的软件为ProteinLynx Global Server 2.1 softwarepackages。
优选地，质谱仪采用的调机及校正标准品为[Glu]-Fibrinopeptide B。
本发明解决其技术问题所采用的技术方案之二是：
三斑海马的水萃取物在制备抗炎药物中的用途，所述水萃取物通过以下方法制备得到：新鲜三斑海马经冷冻干燥后以酒精萃取，酒精萃取后剩下的海马以体积比为0.8～1.2:0.8～1.2的二氯甲烷-甲醇混合液连续萃取若干次，得到的萃取液经浓缩后，以水和乙酸乙酯进行液液相分配萃取若干次，收集水层，再用正丁醇进行若干次分配萃取，收集水层，再除去溶剂，得到所述水萃取物。
本发明所涉及的设备、试剂、工艺、参数等，除有特别说明外，均为常规设备、试剂、工艺、参数等，不再作实施例。
本发明所列举的所有范围包括该范围内的所有点值。
本发明所述“大约”、“约”或“左右”等指的是所述范围或数值的±20％范围内。
本发明中，除有特别说明外，所述方法均在常规环境温度下进行，例如可以为10～30℃。
本技术方案与背景技术相比，它具有如下优点：
本发明提供了一种特殊的液相色谱-电喷雾电离串联质谱法检测三斑海马中牛磺酸和精氨酸的方法，无须经过HPLC管柱前或后衍生化方法即可侦测牛磺酸和精氨酸的含量，具体分析步骤简单易操作；所得到的化合物为海马中提取、分离纯化得到，这对海洋天然产物有效成分的研究具有十分重要的意义。
附图说明
图1为分析得出的牛磺酸和精氨酸的离子层析图(ion chromatograms)；其滞留时间分别为4.51和19.68分钟，牛磺酸的质荷比(m/z)为126[M+H]+而精氨酸的质荷比(m/z)为175[M+H]+。
图2为分析得出的牛磺酸和精氨酸的二次质谱(Ion spectra of productionions(MS2))图，其子离子质荷比分别为108和158。
图3为COX-2的蛋白表达量。
图4为iNOS的蛋白表达量。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
实施例
本实施例采用养殖型三斑海马。将新鲜三斑海马置于95％酒精中保存，取用尾柄肌肉并设计引物(primer)进行基因序列分析，以确定其种属，确认为三斑海马(H.trimaculatus)。养殖成功的养殖型三斑海马(H.trimaculatus)约125只，先养殖约两周，待海马体内残留的药物(如抗生素等)散去后，再按照以下方法进行萃取：
取新鲜三斑海马约1kg经冷冻干燥后以1000mL酒精进行第一次萃取，得到酒精萃取物(Crude extract)(3.2g)；然后剩下的海马以二氯甲烷:甲醇1:1(3000mL)连续萃取三次，其萃取液经减压浓缩后，以水和乙酸乙酯进行液液相分配萃取三次，分别收集乙酸乙酯层和水层；合并每次萃取得到的乙酸乙酯层，得到乙酸乙酯萃取物(2.3g)；高极性的水层再与正丁醇进行三次分配萃取，每次得到的正丁醇层合并，经减压浓缩得到正丁醇萃取物(3.4g)；剩余的水层再经减压浓缩除去溶剂，得到水萃取物(3.1g)。
针对三斑海马萃取得到的酒精萃取物、乙酸乙酯萃取物、正丁醇萃取物和水萃取物，以液相色谱-电喷雾电离串联质谱法(LC-ESI-MS/MS)，测定三斑海马各种萃取物中牛磺酸(Taurine)以及精氨酸(Arginine)含量，具体如下：
液相LC条件：Waters CapLC液相色谱系统；色谱柱Waters Atlantis dC18(3μm，75μm×50μm)；进样量：1μL；柱温：25℃；流动相为溶剂A和溶剂B的混合液，所述溶剂A为0.1％九氟戊酸的水溶液，所述溶剂B为0.1％九氟戊酸的乙腈溶液；采用梯度洗脱程序：流动相中溶剂B的初始比例为5％，随后在25min内由5％上升至60％，随后在5min内降回5％，接着再平衡5min；流动相流速为6μL/min，待进入色谱柱前流速由分流器减至0.3μL/min；
质谱MS条件：Waters Micromass Q-TOF Ultima Global质谱仪(Micromass,Manchester,UK)；质谱仪电离方式设定为+ESI模式(positive ionization mode)，电压4.50kV，脱溶剂温度(desolvation temperature)为290℃，飞行时间检测控制在加速电压9.1kV、锥电压为100V、撞击能量为10eV；质谱扫描范围为m/z 50～500，质谱仪调机及校正标准品为[Glu]-Fibrinopeptide B(Sigma Chemicals；St.Louis MO)，仪器控制及数据收集的软件分别为MassLynx V4.0和ProteinLynx Global Server 2.1 software packages(Waters Corporation,United Kingdom,2005)。
取1μL的牛磺酸(Taurine)和精氨酸(Arginine)的含量液注入系统，结果如图1及图2所示。牛磺酸和精氨酸的滞留时间分别为4.51和19.68分钟，牛磺酸的质荷比(m/z)为126[M+H]+，精氨酸的质荷比(m/z)为175[M+H]+；牛磺酸和精氨酸的子离子质荷比分别为108和158。
根据图1及图2的结果，采用选择反应监测模式(selected reaction model；SRM)，以子离子的离子层析波峰面积比对标准品，进而计算海马萃取物中此两种成分的含量。
表1为SRM模式下最佳的质谱分析参数(Optimized MS parameters for SRMdetermination of underivatized Taurine and L-arginine)。牛磺酸和精氨酸产生子离子所需的最佳撞击能量分别为27eV和33eV。
表1牛磺酸和精氨酸的SRM模式下最佳分析参数
表2为此方法的性能参数(Method performance parameters)。牛磺酸和精氨酸的检测限(limit of detection；LOD)分别为0.313μg/mL和0.174μg/mL，而其定量限(limitof quantification；LOQ)分别为0.625μg/mL和0.435μg/mL。
表2性能参数
a检测限(LOD)定义为信噪比(S/N)为3时对应的每种分析物的浓度
b定量限(LOQ)定义为信噪比(S/N)为10时对应的每种分析物的浓度
表3为此方法的方法学验证数据(Method validation data)。此方法提供良好的准确性，其滞留时间和波峰面积的相对标准偏差值(RSD％)为0.25至3.34之间，且其标准品添加试验提供了90％左右的回收率。
表3方法学验证数据
a保留时间(retention time)
b峰面积(peak area from ion chromatograms)
表4三斑海马各种萃取物中的牛磺酸和精氨酸含量
海马各种萃取物中牛磺酸和精氨酸的含量如表4所示。酒精萃取物(crudeextract)含有少量的精氨酸约0.006mg/g，没有检测到牛磺酸；乙酸乙酯萃取物(EA layerextract)没有检测到牛磺酸和精氨酸；反而是较为亲水的水萃取物(Water layerextract)及正丁醇萃取物(BuOH layer extract)中都有牛磺酸和精氨酸的存在，其中又以水萃取物中的含量较高，分别为6.807mg/g(干重)和0.437mg/g(干重)；水萃取物和正丁醇萃取物中的牛磺酸的总量为8.454mg/g(干重)，此含量高于大部分动物来源的牛磺酸如牛0.4～8.1mg/g(干重)、猪2.4～3.3mg/g(干重)、鸡0.5～7.3mg/g(干重)，海产品0.9～9.2mg/g(干重)。
上述结果显示本发明的萃取流程适合分离提取牛磺酸及精氨酸，特别是水萃取物中二者含量较高，且上述分析方法可以有效检测萃取物中牛磺酸及精氨酸。
本实施例还对养殖型三斑海马的水萃取物的抗炎活性进行了评估。
养殖型三斑海马H.trimaculatus的水萃取物的体外抗炎活性，通过检测该水萃取物对COX-2和iNOS蛋白表达的抑制作用来体现。
RAW264.7细胞接种于10cm培养皿(culture dish)，每孔控制在3×106个细胞，给予lipopolysaccharide(LPS,1.0μg/mL；sigma L2654)，16小时后分别收集细胞及培养基。抗炎实验组中，先加入三斑海马的水萃取物，十分钟后再加入LPS，16小时后分别收集细胞及培养基。
收集RAW264.7细胞，利用iNOS抗体(polyclonal,Transduction Laboratories,Lexington,KY,USA)及COX-2抗体(polyclonal,Cayman,Ann Arbor,MI,USA)，采用westernblot法分析iNOS及COX-2的蛋白表达量。COX-2及iNOS的蛋白表达量分别如图3、图4所示，结果表明，三斑海马的水萃取物具有一定的抑制iNOS和COX-2蛋白表达量的作用。
对比例1
采用液相色谱-电喷雾电离串联质谱法(LC-ESI-MS/MS)检测海马萃取物中的牛磺酸和精氨酸：
液相LC条件：Waters CapLC液相色谱系统；色谱柱Waters Atlantis dC18(3μm，75μm×50μm)；进样量：1μL；柱温：25℃；流动相为甲醇：0.1％甲酸的水溶液＝77：23(v/v)；流动相流速为6μL/min，待进入色谱柱前流速由分流器减至0.3μL/min；
质谱MS条件：Waters Micromass Q-TOF Ultima Global质谱仪(Micromass,Manchester,UK)；质谱仪电离方式设定为+ESI模式(positive ionization mode)，电压3.80kV，脱溶剂温度(desolvation temperature)为150℃，飞行时间检测控制在加速电压9.1kV、锥电压为100V、撞击能量为10eV；质谱扫描范围为m/z 50～500。
在上述检测条件下，无法将海马萃取物中的牛磺酸和精氨酸与其他氨基酸分开。
对比例2
采用反相高效液相色谱法(Reversed-phase high performance liquidchromatography；RP-HPLC)检测海马萃取物中的牛磺酸和精氨酸：
10mg海马萃取物冻干样品以1mL的0.1％三氟乙酸(TFA)溶液(溶剂为水：乙腈＝9:1，v/v)溶解后以0.45μm滤膜过滤(Osmonics Inc.,USA)，制成样品液。
液相条件：Thermo Scientific Surveyor高效液相色谱系统(Waltham,MA,USA)配置photo diode array(PDA)检测器，样品注射量为20L(100μL sample loop,LifeScience,USA)；预过滤分离色谱柱：RP-HPLC Guard Column(4.6×50mm,J.T.Baker,USA)；主要反相色谱柱：C4 column(5micron pore size,4.6×250mm；Restek Co.,USA)；流动相为溶剂A和溶剂B的混合液，溶剂A为0.1％三氟乙酸的水溶液，溶剂B为0.1％三氟乙酸的溶液(溶剂为乙腈：去离子水＝70:30，v/v)；流速：0.5mL/min；采用梯度洗脱程序：流动相中溶剂B的比例在30min内由30％上升至70％；检测波长：220nm。
在上述检测条件下，无法将海马萃取物中的牛磺酸和精氨酸与其他氨基酸分开。
以上所述，仅为本发明较佳实施例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。
Claims (10)
1.一种液相色谱-电喷雾电离串联质谱法检测三斑海马中牛磺酸和精氨酸的方法，其特征在于：包括：
液相条件：Waters CapLC液相色谱系统；色谱柱Waters Atlantis dC18，3μm，75μm×50μm；进样量：1μL；柱温：25℃；流动相为溶剂A和溶剂B的混合液，所述溶剂A为0.1％九氟戊酸的水溶液，所述溶剂B为0.1％九氟戊酸的乙腈溶液；采用梯度洗脱程序：流动相中溶剂B的初始比例为5％，随后在25min内由5％上升至60％，随后在5min内降回5％，接着再平衡5min；流动相流速为6μL/min，待进入色谱柱前流速减至0.3μL/min；
质谱条件：Waters Micromass Q-TOF Ultima Global质谱仪；电离方式：+ESI模式；电压4.50kV，脱溶剂温度290℃；飞行时间检测控制在加速电压9.1kV、锥电压100V、撞击能量10eV；质谱扫描范围为m/z 50～500；选择反应监测模式；以子离子的离子层析波峰面积比对标准品，进而计算三斑海马中牛磺酸和精氨酸的含量。
2.根据权利要求1所述的方法，其特征在于：采用三斑海马的萃取物进行检测；所述萃取方法包括：新鲜三斑海马经冷冻干燥后以酒精萃取，得到酒精萃取物；酒精萃取后剩下的海马以体积比为0.8～1.2:0.8～1.2的二氯甲烷-甲醇混合液连续萃取若干次，得到的萃取液经浓缩后，以水和乙酸乙酯进行液液相分配萃取若干次，分别收集乙酸乙酯层和水层；合并乙酸乙酯层，得到乙酸乙酯萃取物；水层再用正丁醇进行若干次分配萃取，合并正丁醇层，得到正丁醇萃取物；剩余的水层再除去溶剂，得到水萃取物；再以所述酒精萃取物、乙酸乙酯萃取物、正丁醇萃取物和水萃取物进行牛磺酸和精氨酸的检测。
3.根据权利要求1所述的方法，其特征在于：牛磺酸的质荷比为126，牛磺酸的子离子的质荷比为108。
4.根据权利要求1所述的方法，其特征在于：精氨酸的质荷比为175，精氨酸的子离子的质荷比为158。
5.根据权利要求1所述的方法，其特征在于：牛磺酸产生子离子所需的最佳撞击能量为27eV。
6.根据权利要求1所述的方法，其特征在于：精氨酸产生子离子所需的最佳撞击能量为33eV。
7.根据权利要求1所述的方法，其特征在于：仪器控制的软件为MassLynx V4.0。
8.根据权利要求1所述的方法，其特征在于：数据收集的软件为ProteinLynx GlobalServer 2.1software packages。
9.根据权利要求1所述的方法，其特征在于：质谱仪采用的调机及校正标准品为[Glu]-Fibrinopeptide B。
10.三斑海马的水萃取物在制备抗炎药物中的用途，其特征在于：所述水萃取物通过以下方法制备得到：新鲜三斑海马经冷冻干燥后以酒精萃取，酒精萃取后剩下的海马以体积比为0.8～1.2:0.8～1.2的二氯甲烷-甲醇混合液连续萃取若干次，得到的萃取液经浓缩后，以水和乙酸乙酯进行液液相分配萃取若干次，收集水层，再用正丁醇进行若干次分配萃取，收集水层，再除去溶剂，得到所述水萃取物。
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