Post-column hydrogen-deuterium exchange technique to assist in the identification of small organic molecules by mass spectrometry

Emmanuel Eysseric1, Xavier Bellerose1, Jean-Michel Lavoie2, Pedro A. Segura1, *

1 Department of Chemistry, Université de Sherbrooke, 2500 Boulevard de l’Université, Sherbrooke, QC, Canada J1K 2R1

2 Department of Chemical and Biotechnical Engineering, Université de Sherbrooke

* Corresponding author: e-mail: , Tel: 1-819-7922, Fax: 1-819-821-8017

Supplementary material

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Determination of deuteration percentage

The deuteration percentage of trimethoprim was calculated using an algorithm previously published1. Here we present a detailed account of how this algorithm was employed to determine deuteration percentages in the experiments described in the manuscript.

For a small molecule with a formula of CwHxOyNz such as trimethoprim (C14H18O3N4), the main isotopic contributions are 13C (1.1% per carbon atom) and 15N (0.37% per nitrogen atom) whereas 17O, 18O and 2H contributions are negligible.

First, the abundance due to naturally occurring X+1 isotopes (Anatural) is determined with the equation:

Anatural=1.1%×w+(0.37%×z)

Where w and z indicate the number of carbon and nitrogen atoms, respectively. For trimethoprim, Anatural in the M+n peaks is 16.88% of the M+(n-1) peak, therefore this value must be subtracted to obtain the artificial contribution due to HDX. For example, the following data was obtained for trimethoprim using the post-column HDX technique (Table S1).

Table S1. Relative intensities of all the peaks of the isotopic pattern trimethoprim obtained after loop injections using a flow rate of 300 mLmin-1 for the mobile phase (A: 30% of 0.1% FA in H2O, B: 70% of 0.1% FA in ACN) and an addition rate of 50 mLmin-1 of D2O in the QqQMS.

Peak / Ion
m/z / Relative intensity
(%)
M / 291 / 3.59
M+1 / 292 / 24.35
M+2 / 293 / 76.71
M+3 / 294 / 100.00
M+4 / 295 / 40.60
M+5 / 296 / 8.42
M+6 / 297 / 1.13

The corrected relative intensity for the M+1 peak is calculated as follows:

Corr. rel. intensity M+1=rel. intensity M+1-[rel. intensity M×Anatural]

Therefore, the corrected relative intensity for the M+1 of trimethoprim in Table S1 is 23.74 %. The same procedure is applied to the following M+n peaks to obtain Table S2:

Table S2. Corrected relative intensity values for all peaks of the trimethoprim isotopic pattern after post-column HDX.

Peak / Ion
m/z / Relative intensity
(%) / Correction value / Corrected relative intensity
(%)
M / 291 / 3.59 / 0 / 3.59
M+1 / 292 / 24.35 / 0.61 / 23.74
M+2 / 293 / 76.71 / 4.11 / 72.60
M+3 / 294 / 100.00 / 12.95 / 87.05
M+4 / 295 / 40.60 / 16.88 / 23.72
M+5 / 296 / 8.42 / 6.85 / 1.57
M+6 / 297 / 1.13 / 1.42 / -0.29

As it can be observed, this calculation shows that the corrected relative intensity for the M+6 peak is negative, indicating that only 5 deuterium atoms were exchanged (4 labile hydrogens plus the proton adduct). Then, the ratio between each corrected intensity and the sum of all corrected relative intensities is calculated and expressed as a molar percentage. For trimethoprim, the sum all of corrected intensities is 211.98%. Therefore, we obtain the values shown in Table S3:

Table S3. Molar percentage for all peaks of the trimethoprim isotopic pattern after post-column HDX.

Peak / Ion
m/z / Corrected relative intensity
(%) / Molar percentage
(%)
M / 291 / 0.64 / 1.69
M+1 / 292 / 8.34 / 11.20
M+2 / 293 / 47.27 / 34.25
M+3 / 294 / 91.78 / 41.07
M+4 / 295 / 26.14 / 11.19
M+5 / 296 / 0.93 / 0.74

Finally, the deuteration percentage is calculated adding the products of the molar percentage (MP) of each peak by their maximum deuterium fraction (DF). Results are shown in Table S4.

Deuteration percentage=i=1nDFi×MPi

Table S4. Percentage of deuterium for all peaks of the trimethoprim isotopic pattern after post-column HDX.

Peak / Ion
m/z / Molar percentage
(%) / Maximum deuterium fraction / Percentage of deuterium
M / 291 / 1.69 / 0 / 0.00
M+1 / 292 / 11.20 / 0.2 / 2.24
M+2 / 293 / 34.25 / 0.4 / 13.70
M+3 / 294 / 41.07 / 0.6 / 24.64
M+4 / 295 / 11.19 / 0.8 / 8.95
M+5 / 296 / 0.74 / 1 / 0.74

The maximum deuterium fraction is calculated according to the total number of deuterium atoms exchanged. For trimethoprim, the total number is five; therefore, there is a difference of 1/5 between the maximum deuterium fractions of peak having 1 mass unit of difference in the isotopic pattern. The deuteration percentage is calculated by adding the percentage of deuterium of all peaks. For this specific experiment the deuteration percentage was 50.3%.

Generation of theoretical mass spectra

Once the deuteration percentage is calculated, the theoretical mass spectra can be generated using a binomial distribution also known as the Pascal’s triangle. The number of coefficients in a row must be equal to the number of possible configurations. Then, the percentage of 1H is the first coefficient and the percentage of 2H (deuterium) is the second one.

For example, trimethoprim has 4 exchangeable hydrogens plus 1 in positive ionization for a total of 5 exchangeable hydrogens, so 6 possible configurations. The corresponding row is the one where n = 5 and there are 6 coefficients: 1, 5, 10, 10, 5 and 1. The two terms of the distribution here are the calculated percentage of 1H, noted HP and the calculated percentage of 2H, noted DP. The binomial distribution then looks like this:

(HP + DP)5= HP5 + 5 HP4 * DP + 10 HP3 * DP2 + 10 HP2 * DP3 + 5 HP * DP4 + DP5

Each one of the six values represent the 6 possible configurations in the spectrum, from the 0% deuterated form (291) to the 100% deuterated one (296).

From then, a correction is applied because of the contribution of the naturally occurring isotopes, 13C and 15N principally, on the spectra. The contribution is basically the reverse of what was done previously, approximately 17% of the M intensity is added to the M + 1 signal.

Fig. S1. Theoretical mass spectra of trimethoprim without (a) and with (b) the naturally occurring isotope correction. Both spectra were generated using the aforementioned method.

References

(1) McCloskey, J. A. Methods Enzymol. 1990, 193, 329.

Fig. S2. Mass spectrum of trimethoprim after HDX in a QqQMS. Mobile phase flow rate was 300 mL min-1 (A: 30% of 0.1% FA in H2O, B: 70% of 0.1% FA in ACN), D2O addition flow rate was 50 mL min-1 and the mixing device was a mixing tee.


Fig. S3. Representation of the flow direction in different tee connectors shapes: tee connector 90° (a), tee connector 180° (b) and mixing tee (c). Inlet A represents D2O and inlet B the mobile phase.

Fig. S4. Extracted ion chromatogram and experimental and theoretical mass spectra of theophylline after post-column HDX of a SPE extract of spiked river water.

Fig. S5. Extracted ion chromatogram and experimental and theoretical mass spectra of caffeine after post-column HDX of a SPE extract of spiked river water.

Fig. S6. Comparaison of intensities of experimental and theoretical and corrected peaks of tricin at 40% deuteration presented in Fig. 6.

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