Facile synthesis of deuterium-labeled denatonium cation and its application in the quantitative analysis of Bitrex by liquid chromatography-mass spectrometry

Quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) is frequently based on the application of isotopically labeled standards which usually have to be specially synthesized. Although the synthesis of deuterated isotopologues is relatively inexpensive, they are not considered as good internal standards due to the possible deuterium effect on the retention time during LC-MS analysis. We developed a method of deuterium labeling of denatonium benzoate (Bitrex) via H/D exchange of its α-carbon hydrogen atoms in CH2 group situated between carbonyl and quaternary ammonium groups. The proposed strategy is rapid, cost-efficient, and does not require derivatization reagents or further purification. The LC-MS analysis of isotopologues revealed that the introduced deuterons do not undergo back exchange under acidic conditions, and the co-elution of deuterated and non-deuterated forms is observed. The obtained deuterated standard was applied in the quantitative LC-MS analysis of Bitrex in commercially available household products. Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8815-y) contains supplementary material, which is available to authorized users.


Chemicals
In order to estimate pD of the analyzed alkaline solutions, pH was measured on a MP230 pH meter (Mettler-Toledo, Greifensee, Switzerland). pD was calculated according to the equation: pD = pH + 0.4 [1]. NMR analysis NMR spectra were recorded on high-field spectrometer Bruker Avance 500 MHz.
Complete 1 H NMR, 13 C NMR and COSY analysis was performed on each step of denatonium benzoate HDX. Samples were dissolved in CDCl 3 .

Liquid Chromatography -Mass Spectrometry (LC-MS) analysis
The Agilent 1200 system, equipped with an Aeris Peptide XB-C18 column (50 mm × 2.1 mm) 3.6 μm bead diameter was used. The LC system was operated with mobile phase:

Preparation of windscreen washer fluids samples for LC-MS analysis
The samples were prepared by the evaporating of 20 mL of each windscreen washer fluid.
Then the sample was redissolved in H 2 O (1 mL). To the 20 µL of such solution deuterated denatonium benzoate standard (2 µL, 0.5 mg/mL) was added. Then the sample was diluted to 145 µL with water and analyzed by LC-MS. Samples were evaporated both under nitrogen stream and by speedvac. The time required for evaporation was very short for low-volume samples (within minutes) whereas for larger amounts it took around 1 hour.

The computational analysis
The quantification of detected denatonium cations in the prepared samples was performed according to the algorithm described by Mirgorodskaya and co-workers [2]. Briefly, the relative concentrations of non-deuterated sample and isotopically labeled standard can be determined by comparing the isotopic distributions of "natural" and labeled compounds with isotopic pattern measured for analyzed sample containing known amount of deuterated standard. To reduce nonsystematical errors resulting from statistical fluctuations the data were analyzed by least squares method basing on abundancies of 6 isotopic peaks found in labeled and unlabeled compound. The comptutational analysis was peprformed using ORIGIN 8 program (OriginLab Corporation, Northampton, MA, USA)

NMR Analysis
In the 1 H NMR spectrum recorded for non-deuterated denatonium benzoate dissolved in CDCl 3 an intensive signal at 4.97 ppm, corresponding to the α-CH 2 group is present (Fig. 1S A). After the incubation in 1% TEA/D 2 O solution the disappearance of the signal was observed. This clearly indicates that α-C hydrogens were replaced by deuterons (Fig. 1S B). This result confirms that the specific deuteration of the α-C hydrogens takes place in denatonium cation.   Quaternary ammonium compounds containing strong electron-withdrawing group attached to one of the methylene carbon bound to the quaternary nitrogen have been reported to undergo Stevens rearrangement in the presence of a strong base to give rearranged tertiary amines [3].
Additionally it was found by Ng and co-workers [4] that the presence of 2,6-xylycarbamoyl group close to the quaternary nitrogen atom facilitates the loss of a labile proton from the methylene group with subsequent migration of a benzylic group from the quaternary nitrogen         The experimental isotopic patterns for analyzed sample were compared with those obtained according to the computational procedure. The results obtained for the analysis of 1300, 500 and 20 µL of alcoholic thinner are presented below (Fig. 4A-C). Other spectra are presented in supplementary data (Fig. 23S). The same results were obtained when deuterated denatonium benzoate standard for quantitative analysis was directly added to the analyze sample (experiment not shown in experimental section).