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Parameters That Influence MS Quantitation

 

1.      Number of Points Across the Chromatographic Peak

2.      Spray Stability

3.      Peak Shape

4.      Chromatography

5.      Additives

6.      Internal Standard

7.      Mass Resolution

 

  1. Number of Points Across the Chromatographic Peak
    1. 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.
    2. 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.

 

  1. Spray Stability
    1. 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.

 

  1. Peak Shape
    1. 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.

  1. Chromatography
    1. It is true that ms or ms2 can surmount some chromatographic problems, but remember, good chromatography will give you better quantitation and calibration curves.
    2. 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.  
    3. 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.
    4. 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.
    5. Don’t forget about chromatography just because you have a mass spectrometer.

 

  1. Additives
    1. 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.
    2. TFA suppresses MS signal. When you must use TFA, use as little as possible.
    3. Minimize the use of ion pairing reagents. If necessary make sure they are volatile and at low levels.

 

  1. Internal standard
    1. 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..

 

  1. Mass Resolution
    1. 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.
    2. 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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