Chemicals and reagents
Methanol and acetonitrile of LC–MS grade were supplied from Sigma-Aldrich (Steinheim, Germany). Ammonium acetate used as a mobile phase additive was also purchased from Sigma-Aldrich (Steinheim, Germany). 2-pyridinecarbonitrile (98%), 3-pyridinecarbonitrile (98%), 4-pyridinecarbonitrile (98%), propan-2-ol, hydroxylamine hydrochloride (99%), 1-bromodecane and 1-bromo-2-ethylhexane were supplied from Sigma-Aldrich (Steinheim, Germany). NaOH (p.a), were supplied from POCH S.A. (Gliwice, Poland). The LC-grade water (below 1 μS/mL) was prepared by reverse osmosis in a Demiwa 5ROI system from Watek (Ledec nad Sazavou, Czech Republic), followed by double distillation from a quartz apparatus. Only freshly distilled water was used. Standards of N-(2-ethylhexyloxy)pyridine-2-carboximidamide, N-(2-ethylhexyloxy)pyridine-3-carboximidamide, N-(2-ethylhexyloxy)pyridine-4-carboximidamide, N-decyloxypyridine-2-carboximidamide, N-decyloxypyridine-3-carboximidamide and N-decyloxypyridine-4-carboximidamide were synthesised and confirmed by NMR spectroscopy (Bruker Avance II 400 MHz UltraShield Plus)
Synthesis of the standards proceeded in a glass reactor with mechanical stirring at the boiling temperature of propan-2-ol. In the first stage, N-hydroxypyridine-2-, -3- and -4-carboximidamide prepared according to a procedure described by Bernasek (1956), as a solution (0.1 mol in 200 mL propan-2-ol) was heated with sodium hydroxide (0.12 mol in 50 mL water:propan-2-ol solution (2:8, v/v) for 30 min. Then, the mixture of 1-bromodecane or 1-bromo-2-ethylhexane (0.1 mol in 50 mL propan-2-ol) was added dropwise to this mixture, which includes N-hydroxypyridinecarboximidamide and NaOH, and next the whole mixture was heated at 85 °C for 3 h. Reaction products were purified by extraction with chloroform and finally by vacuum distillation. A purity of synthesized compounds was confirmed by NMR spectroscopy (Tables 1, 2).
Analyses were performed using the UltiMate 3000 RSLC LC system (Dionex, Sunnyvale, CA, USA) connected with an API 4000 QTRAP triple quadruple mass spectrometer (AB Sciex, Foster City, CA, USA). Chromatographic separation were done using reverse phase elution with a Spherisorb ODS2 column (50 mm × 4.6 mm I.D.: particle size 5 μm) (Waters, USA). The mass spectrometer was equipped with an electrospray interface operating in positive-ion mode.
Determination of N-alkyloxypyridinecarboximidamines at real synthesis conditions was done using a workstation EasyMax 102 Advanced laboratory reactor with a capacity of 100 mL. The reactor was equipped with reflux cooler, magnetic stirrer bar and temperature sensor. Precise temperature control (±0.1 °C) in the reactor was made possible by solid state thermostat.
The mobile phase used for the sample analysis consisted of 5 mmol L−1 ammonium acetate in a water and methanol mixture at flow rate of 0.6 mL min−1. The gradient was starting at 20% water and 80% methanol and changing linearly to 100% methanol in 2 min, with a final 4.5-min holding period. The duration between subsequent injections was 10 min.
ESI condition: curtain gas 10 psi, nebulizer gas 40 psi, auxiliary gas 40 psi, temperature 400 °C, ion spray voltage 5500 V and collision gas set to medium. Quantifications were performed in multiple reaction monitoring mode (MRM), and the following MRM transitions of [M+H]+ precursor ions → product ions were selected for each analyte: N-(2-ethylhexyloxy)pyridine-2-carboximidamine (t
R = 2.09 min)—m/z 250 → 105 (CE = 39 V), 250 → 120 (CE = 25 V) and 250 → 138 (CE = 23 V); N-(2-ethylhexyloxy)pyridine-3-carboximidamine (t
R = 1.61 min)—m/z 250 → 79 (CE = 61 V), 250 → 105 (CE = 41 V) and 250 → 121 (CE = 29 V); N-(2-ethylhexyloxy)pyridine-4-carboximidamine (t
R = 1.70 min)—m/z 250 → 79 (CE = 49 V), 250 → 121 (CE = 33 V) and 250 → 138 (CE = 27 V); N-decyloxypyridine-2-carboximidamide (t
R = 2.57 min)—m/z 278 → 78 (CE = 59 V), 278 → 96 (CE = 43 V) and 278 → 120 (CE = 27 V); N-decyloxypyridine-3-carboximidamide (t
R = 1.90 min)—m/z 278 → 78 (CE = 59 V), 278 → 96 (CE = 43 V) and 278 → 120 (CE = 27 V); N-decyloxypyridine-4-carboximidamide (t
R = 1.98 min.)—m/z 278 → 79 (CE = 51 V), 278 → 105 (CE = 59 V), 278 → 120 (CE = 33 V) and 278 → 121 (CE = 33 V). Collision energy (CE) was optimized with the “quantitative optimization” function of analyst 1.3.1 or 1.3.2. The monitored fragmentations were selected according to fragmentation pathways of pyridine amidoxime ethers described by Pearse and Jacobsson (1980). The dwell time for mass transition detected the MS/MS multiple reaction monitoring mode (MRM) was set at 50 ms.
Linearity of the calibration was confirmed by analyzing solutions of standards (N-(2-ethylhexyloxy)pyridine-2-carboximidamide, N-(2-ethylhexyloxy)pyridine-3-carboximidamide, N-(2-ethylhexyloxy)pyridine-4-carboximidamide, N-decyloxypyridine-2-carboximidamide, N-decyloxypyridine-3-carboximidamine and N-decyloxypyridine-4-carboximidamide) at different concentrations ranging from 2.5 × 10−5 to 1.0 μg mL−1 [number of points = 16 (n = 3)]. Another determined value was limit of detection (LOD), defined as the concentration that yielded signal-to-noise (S/N) ratios greater than or equal to 3, and limits of quantification (LOQ), defined as the concentration of analyte yielding S/N ratios greater than or equal to 10. Accuracy and precision were carried out with three replicates of three different concentrations low, medium and high quality control samples ranging from 0.2 to 50 ng mL−1 and prepared mixtures containing also substrates of the reaction. Accuracy and precision was determined by injecting a sample with known concentration and calculation of the concentration from the graph and percentage relative standard deviation.
Application of method to monitor N-alkyloxypyridinecarboximidamide at real synthesis conditions
To perform the quantitative analysis of hydrophobic N-alkyloxypyridinecarboximidamides concentration at real synthesis conditions, a series of experiments were carried out under precise conditions. The reactions are done through a workstation EasyMax 102 Advanced laboratory reactor with a capacity of 100 mL. In reaction of synthesis, 1.37 g (0.01 mol) of starting substrate (N-hydroxypyridine-2-, -3- and -4-carboximidamide) dissolved in 100 mL of propan-2-ol was used. Stirring in the reactor was carried out using the magnetic stirrer at 500 rpm and the temperature of reactor was 50 °C. In a further step, 0.40 g (0.01 mol) of sodium hydroxide was added to the reaction mixture. The reaction was run for 15 min with a noticeable change of reaction mixture color to bright yellow, which indicated the occurrence of the reaction and the formation of the sodium salt of the N-hydoxypyridinecarboximidamide. The blank sample was taken before the addition of alkyl bromide (decyl or 2-ethylhexyl bromide) and dissolved in 5 mL of propan-2-ol. In the next step, 0.01 mol of alkyl bromide was added and the mixture was maintained for 120 min at 50 or 80 °C. Samples were taken every 5 min throughout the reaction. Before LC/MS/MS analysis all obtained samples were diluted to the maximum N-alkyloxypyridinecarboximidamide concentration 10 ng mL−1 and next were diluted with 5 mmol L−1 ammonium acetate to obtain a final analyte concentration of 0.25–0.5 ng mL−1. The concentration of the synthesized N-alkyloxypyridinecarboximidamide was calculated from the constructed linear regression equations, and additionally, standard was run before and after stock samples including one control sample and blank control sample.