Determining the Site of Modification … Part 2

Peak matching involves the process of comparing spectral data from a parent or starting material to an unknown compound. (The unknown compound can be referred more specifically as the product, impurity, degradant, metabolite, etc.). The similarities between the data indicate regions that have not changed while the differences indicate regions of change.

The full scan MS data in Part 1 does not offer enough spectral information to eliminate one of the candidate structures. The next step is to examine MS/MS data.

The ESI+ product ion spectra (MS/MS) for both the Parent (m/z 226) and Metabolite (m/z 228) are shown below. The spectra share a common fragment at m/z 121 and differ in the fragments at m/z 134/136 and 149/151.

Based on the fragments shown below, the suspected site of hydrogenation is on the carbonyl for metabolite A.

 

Determining the Site of Modification … Part 2

Peak matching involves the process of comparing spectral data from a parent or starting material to an unknown compound. (The unknown compound can be referred more specifically as the product, impurity, degradant, metabolite, etc.). The similarities between the data indicate regions that have not changed while the differences indicate regions of change.

The full scan MS data in Part 1 does not offer enough spectral information to eliminate one of the candidate structures. The next step is to examine MS/MS data.

The ESI+ product ion spectra (MS/MS) for both the Parent (m/z 226) and Metabolite (m/z 228) are shown below. The spectra share a common fragment at m/z 121 and differ in the fragments at m/z 134/136 and 149/151.

Based on the fragments shown below, the suspected site of hydrogenation is on the carbonyl for metabolite A.

 

Determining the Site of Modification … Part 1

In past blogs, I have ascribed the process of peak matching as an integral part of structure elucidation. In this series, peak matching is demonstrated with the use of MS data.

The scheme below shows a parent compound (2-oxo-N,2-diphenylacetamide) with two possible sites for hydrogenation. The potential modified compounds are 2-hydroxy-N,2-diphenylacetamide and 2-hydroxy-1-phenyl-2-(phenylamino)ethanone labeled A and B, respectively.

The ESI+ full scan MS below belong to the parent compound (top) and the modified compound (bottom). With the given data, can the suspected site of modification be determined?

 

Determining the Site of Modification … Part 1

In past blogs, I have ascribed the process of peak matching as an integral part of structure elucidation. In this series, peak matching is demonstrated with the use of MS data.

The scheme below shows a parent compound (2-oxo-N,2-diphenylacetamide) with two possible sites for hydrogenation. The potential modified compounds are 2-hydroxy-N,2-diphenylacetamide and 2-hydroxy-1-phenyl-2-(phenylamino)ethanone labeled A and B, respectively.

The ESI+ full scan MS below belong to the parent compound (top) and the modified compound (bottom). With the given data, can the suspected site of modification be determined?

 

Using Peak Deconvolution to Extract the 1H Count

When you work on enough NMR datasets, eventually you will run into cases where peaks are overlapping. One approach is to apply a peak deconvolution (or fitting) algorithm in an attempt to separate out the contribution of each overlapping peak.

The 1H NMR spectrum below shows a multiplet (dd) nearly buried by a large water peak at 3.30 ppm.

Direct integration of the multiplet will give a skewed value due to the contribution from the water peak. If the multiplet at 2.87 ppm equates to 1 proton, then how many protons does the multiplet at 3.24 ppm equate to?

The calculation is shown below:

1. Total RA = Relative Area(Water) + Relative Area(dd)

= 134.85 + 13.98 + 13.54 = 162.37

2. Total DA = Deconvoluted Area(Water) + Deconvoluted Area(dd)

= 30077 + 403 = 30480

3. (Deconvoluted Area(dd) / Total DA) = (Relative Area(dd) / Total RA)

  Relative Area(dd) = 403 * 162.37 / 30480 = 2.14 ~ 2 protons

 

Using Peak Deconvolution to Extract the 1H Count

When you work on enough NMR datasets, eventually you will run into cases where peaks are overlapping. One approach is to apply a peak deconvolution (or fitting) algorithm in an attempt to separate out the contribution of each overlapping peak.

The 1H NMR spectrum below shows a multiplet (dd) nearly buried by a large water peak at 3.30 ppm.

Direct integration of the multiplet will give a skewed value due to the contribution from the water peak. If the multiplet at 2.87 ppm equates to 1 proton, then how many protons does the multiplet at 3.24 ppm equate to?

The calculation is shown below:

1. Total RA = Relative Area(Water) + Relative Area(dd)

= 134.85 + 13.98 + 13.54 = 162.37

2. Total DA = Deconvoluted Area(Water) + Deconvoluted Area(dd)

= 30077 + 403 = 30480

3. (Deconvoluted Area(dd) / Total DA) = (Relative Area(dd) / Total RA)

  Relative Area(dd) = 403 * 162.37 / 30480 = 2.14 ~ 2 protons

Distinguishing Impurities … Conclusion

In the blog series entitled Distinguishing Impurities, several types of NMR experiments were outlined to offer some insight into what to be on the look out for. Although there is no set guideline in this process, the best advice I may offer is to keep track of all the solvents, be aware of the possibility for sample degradation or side products, and finally if a signal does not look right, trial and error may be the only safe bet to ensure nothing is overlooked.

Below is a summary of the each part in the series:

Part 1 covered the aspects a 1H NMR whereby 2 singlets seemed out of place in relation to the remaining 1H signals.

Part 2 described some prep work to check for differences in the integrals of the 1H signals.

Part 3 depicted a 1H-13C HSQC with no obvious indicators that any of the 1H signals were impurities.

Part 4 illustrated how a 1H-1H DQF COSY was a bad choice when dealing with 1H singlets.

Part 5 had a 1H-1H TOCSY spectrum lacking any long range correlations with the 1H singlets.

Part 6 explained how the 1H-13C HMBC lacked any correlation trails with the 1H singlets.