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- Number
of Points Across the Chromatographic Peak
- You
need at least 15 to 20 points across a chromatographic peak for
good quantitation. If you
have fewer points you will not be able to describe the peak
adequately and may lose information, for example you may
miss the top of the peak. Also reproducibility is negatively
affected with fewer points and you will observe RSD’s increasing
to unacceptable values.
- One
can increase the number of points across the peak by scanning
faster however data quality is sacrificed. If you increase the
number of points across the peak you must increase your scan
speed, so the time you look at your mass of interest is shorter. A
shorter dwell time negatively affects s/n. Theoretically the s/n
increases with the square root of the increased dwell time.
- Spray
Stability
- Spray
stability influences signal intensity. A wildly fluctuating spray
also affects peak shape. These poorly shaped peaks give very bad
RSD’s and bad calibration curves. For the integrator it is very
difficult to determine the beginning and ending of the peak. Spray
stability is influenced by several parameters:
i.
Poorly mixed solvents.
If you use premixed solvents or buffers, be sure that the solvents are
well mixed before starting the assay. In addition sudden solvent changes
will negatively affect spray stability.
ii.
Old solvents. Unlike cognac, whisky and some wines HPLC solvents
do not age very well. In
time they absorb salts from their glass containers. They can also form
radicals which give spikes and poor spray stability.
iii.
Badly cut capillaries.
Broken or poorly cut capillaries lead to sputtering and spray
instability. Also with a poorly cut capillary it is possible that the
sprays goes off on an angle and is not aligned optimally with the MS
entrance.
iv.
Polyamide Tail. Some
solvents used for HPLC cause the polyamide coating from the capillaries
to weaken and get longer than the fused silica. You can observe this by
looking at the end of the fused silica capillary. If you see a
“tail” hanging off the capillary you should take care of it. This
leads to sputtering and unstable spray. There are two ways to prevent
this 1.) Cut the capillary regularly or 2.) Burn the polyamide coating
away for 1 or 2 cm. Be careful when you burn the capillary. There are
flammable liquids around and the fused silica becomes extremely brittle
and becomes difficult to handle once burned.
- Peak
Shape
- Bad
chromatographic peak shapes are difficult to integrate. Peak
tailing or leading makes it difficult for the integrator to
determine the beginning or end of the peak. Also it is very
difficult to do the integration reproducibly on irregular peaks.
The same applies for split peaks. Try to get nice sharp peaks even
if sensitivity is not required. “Blobs” are not nice to look
at and are difficult to integrate.
- Chromatography
- It
is true that ms or ms2 can surmount some
chromatographic problems, but remember, good chromatography will
give you better quantitation and calibration curves.
- It
is better to separate your peaks with the LC than with the MS. If
you separate them on the column you have more time per component
so you can use longer scan times resulting in better s/n ratios.
Better s/n ratios are easier to integrate and give you better
RSD’s.
- Try
to avoid too many co-eluting peaks. Co-elution might cause
competition effects during the ionization. If compound A
co-elutes with compound B which is much more basic the more
basic compound B will “steal” protons from compound A
and so suppress it making your assay less sensitive.
- Try
to reconstitute the samples in the inorganic solvent. This will
cause the compounds to stack on the front of the column and give
you sharper peaks.
- Don’t
forget about chromatography just because you have a mass
spectrometer.
- Additives
- Use
only volatile modifiers. Nonvolatile buffers, acids and bases will
decrease your sensitivity by a factor 10 to 100, and they can give
unstable sprays.
- TFA
suppresses MS signal. When you must use TFA, use as little as
possible.
- Minimize
the use of ion pairing reagents. If necessary make sure they are
volatile and at low levels.
- Internal
standard
- The
use of an internal standard will enhance the reproducibility and
precision of your data. With mass spectrometry we can use the best
internal standard available, the compound it self.
We can use the stable isotopic labeled compound. These
compounds are chemically the same and behave the same. The labeled
standard should be “far” enough away from the non-labeled to
avoid signal contribution of the abundance of the natural isotopes
to the signal of the internal standard. If the compound and the
standard are not separated adequately by mass, this will result in
quadratic standard curves and sample interference..
- Mass
Resolution
- How
can mass resolution help you? If you have a co-elution of near isobaric
compounds, high resolution can help you separate them,
if the resolution is high enough. If you do MS2 it is
important that you have the possibility of high resolution with
ms1 and ms2 not only in the ms2 mode . You need the high resolution in ms1
for high resolution precursor ion selection. If you don’t have
this capability you can get a mixed product ion spectrum that will
lead you to the wrong conclusions or wrong quan and
calibration curve results.
- Isobaric
compounds are compounds with the same nominal mass but with a
different molecular formula. E.g. N2 and CO both have
the same nominal mass of 28 amu. With a low resolution instrument
these two compounds can not be separated. Actually the exact
mass of N2 is 28.006148 and the exact
mass of CO is 27.99491464 the mass difference is
0.01123336.
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