关于内标和外标_英文
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下一篇 2007-08-10 23:10:28/ 个人分类:ADME/Tox
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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?
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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. 分析测试百科网rv i8wO%UQ] {6u\
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. "How much internal standard should I
add?", this is an important question. It 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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