CN1890568A - 测定饲料中25－羟基胆钙化醇的方法 - Google Patents

测定饲料中25－羟基胆钙化醇的方法 Download PDF

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CN1890568A
CN1890568A CN200480036788.6A CN200480036788A CN1890568A CN 1890568 A CN1890568 A CN 1890568A CN 200480036788 A CN200480036788 A CN 200480036788A CN 1890568 A CN1890568 A CN 1890568A
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劳伦特·德努
理查德·戈斯勒
彼得·霍夫曼
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Abstract

本发明描述了定量测定动物饲料中25－羟基胆钙化醇的方法。该方法如说明书所述包括步骤：向饲料的水分散液中添加定量的质量不同于25－羟基胆钙化醇且极性近似该化合物的内标化合物，例如26，27－六氘－25－羟基胆钙化醇；用叔丁基甲醚萃取该水分散液；以及用HPLC和质谱对萃取液进一步处理。

Description

测定饲料中25-羟基胆钙化醇的方法
本发明涉及定量测定动物饲料中25-羟基胆钙化醇(25-羟基维生素D3)的方法。25-羟基胆钙化醇可作为Hy-DTM(ROCHE VITAMINS AG，Basel，Switzerland)购得，其用作动物饲料的添加剂以改善动物(例如家畜和宠物)的健康状况。考虑到其生理药力以及狭窄的治疗窗口，该化合物的剂量是十分关键的，因此需要可靠的分析手段来监测饲料中该化合物的量及其在饲料中的均匀分布。现有技术描述了各种定量测定血浆中25-羟基胆钙化醇的方法，这些方法基于免疫测定技术(参见W099/67211)或基于使用衍生物或同位素作为内标的HPLC/质谱法(参见Biological &Pharmaceutical Bulletin(2001)，24(7)，738-743)。然而，当用于分析饲料样品时，这些已知方法并不能令人满意。
已有方法均已表明，对饲料样品中的25-羟基胆钙化醇进行分析是困难的，原因在于饲料中存在大量的固态化学物质和生物物质，而血浆则主要由水组成。两类方法是可用的。使用HPLC和UV检测的物理化学方法以及使用样品净化HPLC和放射性标记免疫试剂的免疫化学方法(参见Bruce.W.Hollis，Calcif.Tissue Int.(1996)58：4-5)。另一方法也是费力的并包括分析步骤，其为了定量而使用放射性材料。此方法由以下步骤组成：添加3H-25-羟基胆钙化醇作为内标，用甲醇萃取，在反相SEP-PAK柱上净化样品，在正相SEP-PAK柱上进一步净化，在正相HPLC及最终内部分析反相HPLC上进一步净化。通过闪烁计数3H-25-羟基胆钙化醇来确定总回收率。通过外标法及264nm处的UV检测来进行定量化。由于最终定量是通过UV完成的，因此样品净化过程十分费力。如此复杂的萃取液纯化要求回收率的确定通过使用放射性标记25-羟基胆钙化醇来完成。这两类方法都十分繁琐，操作特性不良且重现性差。
本发明提供了一种新的多步骤但简单的定量测定25-羟基胆钙化醇的方法，该方法可用于动物饲料样品且能得到满意结果。
更具体地，本发明涉及定量测定动物饲料中25-羟基胆钙化醇的方法，该方法包括步骤：
a)将饲料样品分散于水中并向该样品中添加定量的内标化合物，该内标化合物的质量不同于25-羟基胆钙化醇且其极性近似但不同于25-羟基胆钙化醇；
b)用叔丁基甲醚萃取该水分散液；
c)将醚萃取液进行半制备HPLC；
d)收集含25-羟基胆钙化醇和内标化合物的级分(fraction)；
e)将d)中收集的级分或其一部分进行与质谱联用的HPLC；
f)测定25-羟基胆钙化醇以及添加的内标化合物的质谱峰面积；和
g)通过计算所测质谱峰面积来计算25-羟基胆钙化醇的量。
步骤a)中使用的内标化合物是例如25-羟基胆钙化醇的异构体或衍生物或其同位素标记物(例如氘标记同位素)，例如26，27-六氘-25-羟基胆钙化醇(Tetrahedron Lett.Vol.32，No.24，2813-2816(1991))；或25-羟基麦角钙化醇，或1α-羟基胆钙化醇。优选的内标化合物是26，27-六氘-25-羟基胆钙化醇。内标化合物适宜在饲料样品在水中分散或溶解之前以在甲醇中的溶液形式添加。添加的内标化合物的量并不十分关键。适宜地，添加的内标化合物的量可提供基于25-羟基胆钙化醇约0.05m至约等摩尔的浓度。然后，在步骤b)中将饲料样品的水分散液或溶液用约1-10倍量的叔丁基甲醚萃取，优选伴以超声波处理。通过从步骤b)得到的萃取液蒸发出有机溶剂来完成步骤c)的半制备HPLC，适宜地在排除氧的条件下在硅胶上进行，使用非极性溶剂例如脂族C5-C8烃，例如异辛烷或这样的溶剂与其它极性溶剂的混合物，极性溶剂例如是低级烷醇(例如异丙醇)和/或酯(例如乙酸乙酯)。优选的半制备HPLC体系是硅胶以及体积比约为1∶10∶89的异丙醇、乙酸乙酯和异辛烷混合物。根据步骤e)的HPLC分析适宜在非极性固定相(例如改性的硅胶)的柱上用极性溶剂(例如水或低级烷醇)进行。本文所用术语“改性硅胶”表示反相硅胶，例如以C18烃片断醚化的硅胶，例如由Thermo Hypersil-Keystone，Runcom，UK提供的Aquasil C18。
根据步骤g)的质谱测定的样品中25-羟基胆钙化醇的量由下列方程计算：
Figure A20048003678800052
其中，RF＝响应因子；RRF＝相对响应因子；ISD＝内标溶液；HD＝25-羟基胆钙化醇；c＝浓度[ng/ml]。
用25-羟基胆钙化醇和26，27-六氘-25-羟基胆钙化醇在甲醇∶水(70∶30)溶液中的浓度均为约5ng/ml的溶液来确定相对响应因子(RRF)。
通过以下实施例进一步描述本发明。
实施例
A.萃取：
称量10g饲料样品(包含28.6％大豆、3％鱼粉、2％大豆油、57.3％玉米、2％玉米淀粉、2％木素磺化盐、3.1％大米、2％矿物混合料的混合物)置于锥形瓶中。向其中添加约500ng 26，27-六氘-25-羟基胆钙化醇(0.01ml的2.5mg 26，27-六氘-25-羟基胆钙化醇在50ml甲醇中的溶液)和60ml水，将所得浆液在50℃下在超声波中处理10分钟。然后，添加40ml叔丁基甲醚，将混合物剧烈振荡5分钟，再超声波处理5分钟并离心。分离出10ml有机上层清液并在排除氧的条件下蒸发。
B.半制备HPLC：
将残余物溶解于2ml的异丙醇∶乙酸乙酯∶异辛烷(1∶10∶89)的流动相中，离心处理，并将上层清液的100μl部分注射入半制备HPLC柱，该柱为Hypersil Si60，3μm，120，150×4.6mm(Shandon)。流率为1.0ml/min。收集14-16分钟之间的级分(通过在开始前注射混合标准溶液来检验级分分离)，在50℃下在氮蒸汽中蒸发。使用超声波浴使残余物溶解于0.7ml甲醇中。然后，添加0.3ml水，之后将该溶液注射入与质谱联用的分析HPLC柱中。
C.分析HPLC：
通过与质量比检测器联用的色谱系统来进行分析HPLC。质量比检测器之前的色谱系统由捕集柱(其上浓缩待测定物质)以及用于分离的分析柱组成。
装置示意图见图4。在图4中，“TC”代表捕集柱，“AC”代表分析柱，“MSD”代表质量比检测器。“A”和“B”表示不同操作模式下色谱流动相的容器(receptacle)。
在捕集柱(TC)中，固定相为Aquasil C18，3μm，2.0×10mm。在分析柱(AC)中，固定相为Aquasil C18，3μm，3.0×150mm。流动相为水(含0.05％HCOOH)和甲醇/水(含0.05％HCOOH)梯度溶液。该系统的工作参数如下：
流率：        泵1：0.6ml/min
              泵2：0.7ml/min
注射体积：90μl
注射器温度：5℃
柱温：40℃
停留时间：约4min
色谱法根据下表1所示的方式进行：
表1
  柱切换系统   捕集柱   分析柱
时间 位置 时间   流动相1) 时间   流动相1)
  0-1.65 A   0.00   60％B2   调节
  0.00-1.00   ＞85％B2   负载浓缩   1-1.65   90％B1   调节
  1.00-1.65   85％B2   洗涤
  1.65-2.20 B   1.65-2.20   90％B1   转移前向冲洗   1.65-2.20   90％B1   色谱法开始
2.20-12.00 A 2.20-2.50 85％B2 洗涤 2.20-6.40 90％B1 分离
  2.50-2.60  ＞100％B2   洗涤   6.40-6.50   ＞100％   洗涤
  2.60-9.00  100％B2   洗涤   6.50-9.00   100％B1   洗涤
  9.00-9.10  ＞60％B2   9.00-9.10   ＞90％B1   洗涤，调节
  9.10-12.00  60％B2   调节   9.10-12.00   90％B1   洗涤，调节
1)＞＝梯度(流动相组成的变化)
质量比检测器(MSD)的参数如下：
检测器：Agilent 1946C LC/MSD SL单四极质量比检测器
离子化技术：APCI(大气压化学离子化)
捕获方式：SIM(选择性离子监测)
极性：正
喷射与干燥气体：99.999％的氮气(质量N50)
干燥气体流量：9.5L/min
喷雾器气体压力：50psig
干燥气体温度：225℃
蒸发器温度：250℃
毛细管电压：3000V(Vcap＝离子化电压)
电晕电流：10μA
增益：1.5
SIM参数：
  离子   m/z(M+H)+   粉碎器[V]   停留时间[msec]   相对停留时间[％]
  HyD-H2O   383.3   140   226   30
  D6-HyD-H2OISD 389.3 140 226 30
  HyD   401.3   90   151   20
  D6-HyDISD 407.3 90 151 20
使用上述装置及操作方式，分析标准溶液、空白饲料样品(不含25-羟基胆钙化醇)和普通饲料样品。标准溶液如下制备：
1.25-羟基胆钙化醇
将2.5mg 25-羟基维生素D3溶于50ml甲醇。用甲醇稀释2ml该溶液至200ml以得到浓度为500ng/ml的溶液。
2.d6-25-羟基胆钙化醇(内标)
将2.5mg d6-25-羟基胆钙化醇溶于50ml甲醇。用甲醇稀释2ml该溶液至200ml以得到浓度为500ng/ml的溶液。
3.用甲醇∶水(70∶30)将25-羟基胆钙化醇溶液(1中)与d6-25-羟基胆钙化醇溶液(2中)各1ml稀释至100ml，得到羟基化胆钙化醇浓度为5ng/ml的溶液。
用上面A中所述类似的方法分析空白饲料样品。
标准溶液、空白饲料样品和普通饲料样品的萃取离子色谱图如图1-3所示。通过前面给出的方程计算25-羟基胆钙化醇的量。

Claims (9)

1.定量测定动物饲料中25-羟基胆钙化醇的方法，所述方法包括步骤：
a)将饲料样品分散于水中并向该样品中添加定量的内标化合物，所述内标化合物的质量不同于25-羟基胆钙化醇且其极性近似但不同于25-羟基胆钙化醇；
b)用叔丁基甲醚萃取该水分散液；
c)将醚萃取液进行半制备HPLC；
d)收集含25-羟基胆钙化醇和内标化合物的级分；
e)将d)中收集的级分或其一部分进行与质谱联用的HPLC；
f)测定25-羟基胆钙化醇以及添加的内标化合物的质谱峰面积；和
g)通过计算所测质谱峰面积来计算25-羟基胆钙化醇的量。
2.如权利要求1的方法，其中所述内标化合物为26，27-六氘-25-羟基胆钙化醇、25-羟基麦角钙化醇或1α-羟基胆钙化醇。
3.如权利要求2的方法，其中所述内标化合物为26，27-六氘-25-羟基胆钙化醇。
4.如权利要求1-3中任何一项的方法，其中所述半制备HPLC在作为固定相的硅胶及作为流动相的异丙醇、乙酸乙酯、异辛烷混合物上进行。
5.如权利要求4的方法，其中所述流动相为体积比1∶10∶89的异丙醇、乙酸乙酯与异辛烷。
6.如权利要求4或5的方法，其中所述固定相为3μm的Hypersil Si60。
7.如权利要求1-6中任何一项的方法，其中所述分析HPLC是在包括其上浓缩待测定物质的捕集柱和用于分离的分析柱的色谱系统中进行的。
8.如权利要求4的方法，其中所述分析HPLC中的固定相为改性硅胶，例如3μm的Aquasil C18。
9.如权利要求7或8的方法，其中所述流动相为含0.05vol％蚁酸的水和含0.05vol％蚁酸的甲醇的梯度溶液。
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