内标法及外标法方法、原理、优缺点

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An internal standard should be used when performing MS quantitation. An

appropriate internal standard will control for extraction, HPLC injection and

ionization variability. In a complex matrix it is not uncommon for two different

standard levels in SRM integrated plots, at the lower end of the standard curve,

to give nearly an identical response. It is only when an internal standard is

used that the two points can be differentiated. Some researchers attempt to

prepare standard curves and run samples without an internal standard and find

moderate success. Often without an internal standard % RSDs of replicates

can be as high as 20%. Using an internal standard the % RSDs can be brought

down to approximately 2%. We run triplicates at each level of our standard

curve.

How do I choose an internal standard?

The best internal standard is an isotopically labeled version of the molecule

you want to quantify. An isotopically labeled internal standard will have a

similar extraction recovery, ionization response in ESI mass spectrometry, and

a similar chromatographic retention time. If you are performing non-clinical PK

quantitation it may be difficult to justify such a standard since a special

synthesis of an isotopically labeled standard can be expensive and time

consuming. Often if you are working with medicinal chemists they will have a

library of compound analogs that can be used as internal standards. These

analogs were made in the evolution of the compound to be tested and will be

similar to the compound to be quantified and more importantly will be slightly

different by parent mass. Try to avoid using de-methylated (-14) or

hydroxylated (+16) analogs as internal standards since these are the most

common mass shifts observed in naturally occuring metabolites of the parent

compound. A common internal standard is a chlorinated version of the parent

molecule. A chlorinated version of the parent molecule will commonly have a

similar chromatographic retention time which is an important characteristic of

an internal standard. We have found that one of the most important

characteristics of an internal standard is that it co-elutes with the compound to

be quantified.

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How do I use an internal standard?

First of all an internal standard should be added at the beginning of the sample

work-up, typically before the plasma crash or solid phase extraction. The

internal standard should be added at the same level in every sample including

the standards. An internal standard should give a reliable MS response. Care

should be taken that the amount of the internal standard is well above the limit

of quantitation but not so high as to suppress the ionization of the analyte.

pays to know roughly how much compound is in your sample. This can be

accomplished by making trial analyses of an early, middle and late time point

with perhaps one or two standard points. This information will be very valuable

when building an appropriate standard curve and in knowing how much

internal standard to add. If you were trying to quantify samples in the range of

100 fg to 25 pg and the limit of detection was 100 fg you might add 5 to 10 pg

of internal standard to every sample. A good rule of thumb is to target the

internal standard to the lower 1/3 of the working standard curve. This is a

range that will give a comfortable response without interfering with the

ionization of the analyte.

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中文：

什么叫

< br>内标

法

?

怎样选择内标物

?

word

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.

内标法是一种间接或相对的校准 方法。

在

分析测定

样品中某组分

含量

时，

加入一

种内 标物质以校谁和消除出于操作条件的波动而对分析结果产生的影响，

以提高

分析结果的准确度。

内标法在气相

色谱定量

分析中是一种重要的技术。

使用内标法时，< /p>

在样品中加入

一定量的

标准

物质，

它可被色谱拄所分离，

又不受试样中其它组分 峰的干扰，

只

要测定内标物和待测组分的峰面积与相对响应值，

即可求出待测组分在样品中的

百分含量。

采用内标法定量时，

内标物的选择是一项十分重要的工作。

理 想地说，

内标物应当是一个能得到纯样的己知化合物，

这样它能 以准确、

已知的量加到样

品中去，它应当和被分析的样品组分有 基本相同或尽可能一致的物理

化学

性质

(

如化学结构、极性、挥发度及在溶剂中的溶解度等

)

、色谱行为和响应特征，最

好是被分析物质的一个同系物。

当然，

在色谱分析条什下，

内标物必须能与样品

中各组分充分分离。

需要指出的是，

在少数 情况下，

分析人员可能比较关心化台

物在一个复杂过程中所得到 的回收率，

此时，

他可以使用一种在这种过程中很容

来测定感兴趣化合物的百分回收率，

而不必遵循

以上所说的选择原则。

在使用内标法定量时，有哪些因素会影响内标和被测组分的峰高或峰面积的比

值

?

影响内标和被测组分峰高或 峰面积比值的因素主要有化学方面的、

色谱方面的和

仪器方面的 三类。

由化学方面的原因产生的面积比的变化常常在分析重复 样品时出现。

化学方面的因素包括：

1

、内标物在样品里混合不好；

2

、内标物和样品组分之间发生反应，

3

、内标物纯度可变等。