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1、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 stan
2、dard 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 % R
3、SDs 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 standardThe best internal standard is an isotopically labeled version of the molecule you want to
4、 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
5、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 s
6、imilar 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 comp
7、ound. 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 charac
8、teristics of an internal standard is that it co-elutes with the compound to be quantified.How do I use an internal standardFirst of all an in ter nal sta ndard should be added at the beg inning of the sample work-up, typically before the plasma crash or solid phase extrac tion. The in ter nal sta nd
9、ard should be added at the same level in every sample in clud ing the sta ndards. An in ter nal sta ndard should give a reliable MS resp on se. Care should be take n that the amo unt of the in ter nal sta ndard is well above the limit of qua ntita tion but not so high as to suppress the ionization o
10、f the an alyte. How much in ter nal sta ndard should I add, this is an importa nt ques tion. It pays to know roughly how much compo und is in your sample. This can be accomplished by making trial an alyses of an early, middle and late 廿me point with perhaps one or two sta ndard poin ts. This inf orm
11、a tion will be very valuable whe n build ing an appropriate sta ndard curve and in knowing how much in ter nal sta ndard to add. If you were try ing to qua ntify samples in the range of 100 fg to 25 pg and the limit of detec tion was 100 fg you might add 5 to 10 pg of in ter nal sta ndard to every s
12、ample. A good rule of thumb is to target the in ter nal sta ndard to the lower 1/3 of the work ing sta ndard curve. This is a range that will give a comfortable resp onse without in terferi ng with the ion iza tion of the an alyte.中文:什么叫内标法怎样选择内标物 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
13、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，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
14、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 易被完全回收的化台物作内标，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。对于一
15、个比较成熟的方法来说，色谱方面的问题发生的可能性更大一些，色谱上 常见的一些问题（如渗漏）对绝对面积的影响比较大，对面积比的影响则要小一些， 但如果绝对面积的变化已大到足以使面积比发生显著变化的程度，那么一定有某 个重要的色谱问题存在，比如进样量改变太大，样品组分浓度和内标浓度之间有 很大的差别，检测器非线性等。进样量应足够小并保持不变，这样才不致于造成 检测器和积分装置饱和。如果认为方法比较可靠，而色谱固看来也是正常的话， 应着重检查积分装置和设置、斜率和峰宽定位。对积分装置发生怀疑的最有力的 证据是：面积比可变，而峰高比保持相对恒定，在制作内标标准曲线时应注意什么在用内标法做色谱定量分析时
16、，先配制一定重量比的被测组分和内标样品的混合 物做色谱分析，测量峰面积，做重量比和面积比的关系曲线，此曲线即为标准曲 线。在实际样品分析时所采用的色谱条件应尽可能与制作标准曲线时所用的条件 一致，因此，在制作标准曲线时，不仅要注明色谱条件(如固定相、柱温、载气 流速等)，还应注明进样体积和内标物浓度。在制作内标标准曲线时，各点并不 完全落在直线上，此时应求出面积比和重量比的比值与其平均位的标准偏差，在 使用过程中应定期进行单点校正，若所得值与平均值的偏差小于2，曲线仍可使 用，若大于2，则应重作曲线，如果曲线在铰短时期内即产生变动，则不宜使用 内标法定量。外标法用待测组分的纯品作对照物质，以对照物质和样品中待测组分的响应信号相比较 进行定量的方法称为外标法。此法可分为工作曲线法及外标一点法等。工作曲线 法是用对照物质配制一系列浓度的对照品溶液确定工作曲线，求出斜率、截距。 在完全相同的条件下，准确进样与对照品溶液相同体积的样品溶液，根据待测组 分的信号，从标准曲线上查出其浓度，或用回归方程
