Deuterium Exchange Analysis for Improved Structural Elucidation with the 5975-SMB GC-MS with Cold EI

Aviv Amirav, Professor of Chemistry at Tel Aviv University and Director - Aviv Analytical

Introduction

Hydrogen-Deuterium exchange (also called H-D or H/D exchange) is a chemical reaction in which hydrogen atoms that are bound to nitrogen or oxygen atoms (possibly sulfur) are exchanged with deuterium atoms of certain deuterated solvents such as deuterated methanol (CD3OD or CH3OD) or heavy water (D2O). Deuterium exchange reactions followed by mass spectrometry analysis provide valuable structural information since these reactions are usually selective: only hydrogen atoms that are bound to nitrogen or oxygen atoms (such as in amines or alcohols) are exchanged with deuterium while other hydrogen atoms are unaffected. Consequently, the mass shift of the molecular ion reveals the number of OH and NH bonds in the explored compound which helps in its structural elucidation. Currently, deuterium exchange experiments are limited to NMR and LC-MS and it is practically ignored in GC-MS since:  

1. most of the small compounds that are amenable for GC-MS analysis can be fully identified by the available extensive EI libraries.
2. large compounds on the GC-MS scale are often incompatible with GC-MS analysis and/or do not exhibit molecular ions in their EI mass spectra.
3. While the compound of interest may exchange its labile hydrogen atoms with deuterium, reversed exchange may occur at the GC liner, long column and particularly at the ion source metal surfaces that quickly react with ever-present water in the mass spectrometer vacuum chamber.

We found that the use of GC-MS with Cold EI uniquely enables the monitoring of deuterium exchange reactions and thus with it deuterium exchange can serve as an additional effective tool for improved structure elucidation of unknown compounds.                       

Keep reading for more information on deuterium exchange monitoring using the Aviv Analytical 5975-SMB GC-MS with Cold EI, including two analysis examples of Quinine and 17β-estradiol that serve for the validation of their structure and number of labile OH hydrogen atoms.

Background

Our first exploration of deuterium exchange using GC-MS with Cold EI is described in the paper of S. Dagan and A. Amirav "Cluster Chemical Ionization and Deuterium Exchange Mass Spectrometry in Supersonic Molecular Beams". J. Am. Soc. Mass. Spectrom., 7, 550-558 (1996). In these experiments CH3OD or CD3OD or heavy water was used and loaded instead of the methanol in the cluster CI vial. In this mode of operation the reaction zone length had to be extended to 10-20 cm for full deuterium exchange efficiency, which makes its usage inconvenient. A much simpler way to obtain deuterium exchange data is to dissolve the sample in a deuterated methanol solvent and inject it. Back exchange reactions can occur but as demonstrated below approximately 60% deuterium exchange can be achieved with Cold EI which is enough for the conclusion about the number of labile hydrogen atoms in a given sample compound. The experiments below were performed with Quinine (C20H24N2O2) which is an important anti malaria drug which also possesses other therapeutic uses and 17β-estradiol (C18H24O2) which is an estrogenic sex hormone (steroid). This article explores deuterium exchange as a viable further benefit of GC-MS with Cold EI, adding to the already long list of identification related benefits which make it the ultimate identification GC-MS system.

      

Analytical conditions summary

System: 5975-SMB GC-MS with Cold EI system of Aviv Analytical, based on the combination of an Agilent 5975 MSD with the Aviv Analytical supersonic molecular beam interface and its unique fly-through ion source. 

Sample and solvents: Sigma-Aldrich samples that were dissolved either in methanol or in 99.8% D content CD3OD (Cambridge Isotopes) at about 300 ppm.       

Injection: 1 µL at 250ºC with injection split ratio of 10 for having ~30 ng sample on-column.   

Column: 15 m length, 0.25 mm ID, 0.25µ film DB-5MS UI     

He column flow rate: 8 ml/min.    

Oven: 140ºC followed by 20ºC/min to 300ºC and wait for 2 min for total time of 10 min. 

Cold EI Source: 12 mA emission, 70 eV electron energy, 52 ml/min He makeup flow.  

SMB transferline temperature: 250ºC.  

5975 mass range: 50-500 amu at about 3.2 Hz scanning frequency.  

Deuterium Exchange Results

In Figure 1 we show deuterium exchange analysis of Quinine with the Aviv Analytical 5975-SMB GC-MS with Cold EI. The upper trace shows the generated total ion mass chromatogram that shows only one peak of Quinine at 6.2 min. The middle trace shows the Cold EI mass spectrum of Quinine obtained when is was dissolved in methanol while the bottom trace shows the Quinine Cold EI mass spectrum obtained when it was dissolved in deuterated methanol (methanol-D4 or CD3OD). As demonstrated, Quinine (C20H24N2O2) which is a polar drug with one free OH group can be easily analyzed by the 5975-SMB GC-MS with Cold EI. Note that the Cold EI mass spectrum exhibits a molecular ion with 26% relative abundance while in the NIST library its molecular ion is either missing or at below 1% relative abundance in one of the replica MS. This provision of trustworthy molecular ions is an important feature of Cold EI which by itself uniquely enables the monitoring of deuterium exchange in Quinine. The bottom trace of Figure 1 demonstrates effective deuterium exchange in which the molecular ion mass is increased by one amu from m/z=324 into m/z=325. In Figure 2 we show the Quinine Cold EI MS zoomed around the m/z values of the Quinine molecular ions (isotopomers). The upper trace shows the Quinine Cold EI mass spectrum obtained when it was dissolved in deuterated methanol (methanol-D4 or CD3OD) while the bottom trace shows the Cold EI mass spectrum of Quinine when it was dissolved in regular methanol (CH3OH). Evidently, the deuterium exchange is incomplete and only about 60% of the hydrogen atoms of the OH are exchanged (or higher % was exchanged and than back exchanged into hydrogen at the injector liner and/or column). However, clearly this is sufficient to conclude that Quinine has one and only one OH groups as shown in its structure (included in Figure 1). We note that the deuterium exchange mass spectra contain further structural information in the fragments mass spectral peaks. For example, the m/z= 189 fragment includes an OH group as concluded from the increased m/z value of this peak into 190 in the deuterium exchange experiment while the major fragment ion with m/z= 136 clearly has no OH group in it. We note that in the evaluation of deuterium exchange data one must consider natural isotope abundances which for Quinine (C20H24N2O2) implies a (M+1)/M ratio of 22.72% (325 Da/324 Da).

Figure 1. Quinine analysis with the Aviv Analytical 5975-SMB GC-MS with Cold EI. The upper trace shows the generated total ion mass chromatogram which exhibits only one peak of Quinine at 6.2 min. The middle trace shows the Cold EI mass spectrum of Quinine that was obtained when it was dissolved in methanol while the bottom trace shows the Quinine Cold EI mass spectrum obtained when it was dissolved in deuterated methanol (CD3OD).

Figure 2. Cold EI mass spectra of Quinine obtained with the Aviv Analytical 5975-SMB GC-MS with Cold EI, zoomed around the m/z values of the Quinine molecular ions (isotopomers). The upper trace shows the Quinine Cold EI mass spectrum obtained when it was dissolved in deuterated methanol (CD3OD) while the bottom trace shows the Cold EI mass spectrum of Quinine when is was dissolved in regular methanol (CH3OH). 

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In order to further explore the effectiveness and value of deuterium exchange we tested it also with 17β-estradiol (C18H24O2) as shown in Figure 3. In Figure 3 the upper trace shows the generated total ion mass chromatogram with only one major peak of 17β-estradiol at 5.2 min. The middle trace shows the Cold EI mass spectrum of 17β-estradiol obtained when it was dissolved in deuterated methanol (CD3OD) while the bottom trace zoom on this Cold EI mass spectrum near the m/z range values 269-279 of its molecular ions. As demonstrated, deuterium exchange is effective with 17β-estradiol and the most abundant molecular ion is at m/z=274 instead of 272. While the process of deuterium exchange is incomplete it is sufficiently effective to clearly indicate that 17β-estradiol as its name implies (diol) has two OH groups that can exchange their labile hydrogen atoms into deuterium atoms while all other hydrogen atoms are passive and do not participate in the deuterium exchange process.  

Figure 3. Deuterium exchange analysis of 17β-estradiol (C18H24O2) with the Aviv Analytical 5975-SMB GC-MS with Cold EI. The upper trace shows the generated total ion mass chromatogram with only one major peak of 17β-estradiol at 5.2 min. The middle trace shows the Cold EI mass spectrum of 17β-estradiol obtained when it was dissolved in deuterated methanol (CD3OD) while the bottom trace shows a zoomed view of this spectrum around 269-279Da containing the 17β-estradiol's molecular ions.

  

Conclusions

As demonstrated with Quinine and 17β-estradiol, the Aviv Analytical 5975-SMB GC-MS with Cold EI uniquely enables the use of deuterium exchange for further improved structural elucidation of unknown compounds via the determination of the number of labile hydrogen atoms that are bound to oxygen and/or nitrogen atoms. Furthermore, additional structural information can be asserted via the deuterium exchange induced mass increase of fragment mass spectral peaks.
    

While deuterium exchange investigations by GC-MS are very rare, GC-MS with Cold EI uniquely enables it in view of: 

1. The range of compounds amenable with GC-MS with Cold EI for analysis is significantly increased.
2. All compounds practically exhibit abundant molecular ions in Cold EI. 
3. Reversed hydrogen exchange at the GC liner and column is minimized with the use of shorter columns and particularly with the use of high column flow rates and it is fully eliminated at the Cold EI fly-through ion source. 

Thus, Cold EI can provide further structural information on the number of OH and NH or NH2 in the sample compound via easy to employ and use deuterium exchange experiments of samples in deuterated methanol solvent, adding another viable option that further improves the power of GC-MS with Cold EI in sample identification and structural elucidation.

You can find more on the enhanced identification capabilities of GC-MS with Cold-EI in the following related articles:

Enhancing the Identification Capabilities of EI GC-MS - How Quadrupole GC-MS can compete with High Resolution TOF

The Effects of Enhanced Molecular Ions on NIST's Identification Probability

TAMI - Molecule Identification Software