Quantitative analysis of amphiphilic N-alkyloxypyridinecarboximidamide by liquid chromatography–tandem mass spectrometry

LC–MS/MS method to determine hydrophobic N-alkyloxy substituted amidines: N-(2-ethylhexyloxy)pyridine-2-carboximidamide, N-(2-ethylhexyloxy)pyridine-3-carboximidamide, N-(2-ethylhexyloxy)pyridine-4-carboximidamide, N-decyloxy pyridine-2-carboximidamide, N-decyloxypyridine-3-carboximidamide and N-decyloxypyridine-4-carboximidamide was developed and validated in terms of linearity, precision and accuracy. The developed method was successfully applied to monitor and control the synthesis process. The experimental data points indicated that the straight chain alkyl bromide reacted most rapidly than branched alkyl bromide and the enhancement of the reaction efficiency strongly depended on reaction temperature.

Introduction N-substituted amidines, similar to well known simple amidoximes (Abele et al. 2003), have already been used as antimicrobial (Hall et al. 1998;Boykin et al. 1996), insecticidal (Paul 1981), herbicidal or plant growth regulatory agents (Farge et al. 1978(Farge et al. , 1980 as well as DNA photo-cleavage agents (Karamtzioti et al. 2015) reactivators of nerve agent and pesticide poisoning (Kliachyna et al. 2014) and as regulator of blood pressure (Izawa et al. 1993). The most important key issues for the preparation of such compounds are a complexing nature of the N-hydroxypyridine-2-or -4-carboximidamide, which can coordinate to the divalent metal ions mainly through their pyridine or amine and imine nitrogen atom forming fiveor six-membered chelate rings (Salonen et al. 2008;Salonen 2010;Konidaris et al. 2011;Coropceanu et al. 2014;Nandy et al. 2013). Preparation of simple N-hydroxyimidamides through an O-alkylation reaction has been described frequently (Izawa et al. 1993;Michaelis 1891;Eloy and Lenaers 1962). The alkylation is the process of introducing an alkyl group in the form of a carbocation, carbanion, or an alkyl radical into a molecule of an organic compound by a substitution reaction. In case of N-alkyloxypyridine-2-or -4-carboximidamide, the reaction mechanism consists of two stages (Fig. 1). In the first step sodium 1-amino-1-(2-, 3-or -4-pyridyl)imineoxide is formed by reaction of an appropriate N-hydroxypyridinecarboximidamide with strong base (NaOH, NaOR). The oxygen atom of the created organic sodium salts is thus highly activated and may act as a nucleophile. In the second step, the alkyl halide is attacked by the nucleophile according S N 2 mechanism to form the final product, N-alkyloxypyridinecarboximidamide.
Elimination is always in competition with the substitution reaction and it may occur as unwanted side reactions. A common cause of the by-product formation, beyond alkyl halide structure, type of solvent, polarizability and basicity of nucleophile and type of leaving group, are a too long reaction time and too high temperature. Therefore, a quantitative analysis of the reaction enabling the choice of the optimal parameters is important for the design of an efficient method to synthesize hydrophobic N-alkyloxypyridinecarboximidamides containing decyloxy or 2-ethylhexyloxy group. The GC/MS method is the most commonly used method to determine the progress of N-substituted amidines synthesis (Mijin et al. 2004); however, the application of this method to analysis of compounds dissolved in water or a mixture of water with other diluent requires a modification of the simple preparation such as an extraction or diluents evaporation. Recent development and advancement in analytical technologies has emerged with more sophisticated hyphenated techniques. Among these, one of the most interesting is LC-MS/MS, which allows quantitatively analyze the different analytes in various matrices due to the inherent specificity and sensitivity. LC-MS/MS analyses depend on the use of triple quadrupole mass spectrometers operating in the MRM (multi reaction monitoring) mode (Bijlsma et al. 2009;Boleda et al. 2007;Petrovic et al. 2010). This mode of acquisition provides good sensitivity and selectivity and guarantees reliability of results by recording at least two or more specific SRM (selected reaction monitoring) transitions for each target analyte (Pozo et al. 2006;Jak et al. 2015). LC-MS/MS can be used for quantitative simultaneous analysis of organic compounds even from aqueous alcohol solution; therefore this instrument is the most frequently selected for environmental analysis.
The aim of the current study was to develop a simple and precise analytical method for the quantification of hydrophobic N-alkyloxypyridinecarboximidamides using LC-MS/MS technique directly from synthesis solutions.

Standards synthesis
Synthesis of the standards proceeded in a glass reactor with mechanical stirring at the boiling temperature of propan-2ol. In the first stage, N-hydroxypyridine-2-, -3-and -4carboximidamide 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 Nhydroxypyridinecarboximidamide 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).

Equipments
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 9 4.6 mm I.D.: particle size 5 lm) (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.

LC-MS/MS analysis
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

Method validation
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 9 10 -5 to 1.0 lg mL -1 [number of points = 16 (n = 3)]. Another determined value was limit of detection (LOD), defined as the concentration that yielded signal-tonoise (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 Nalkyloxypyridinecarboximidamide at real synthesis conditions
To perform the quantitative analysis of hydrophobic Nalkyloxypyridinecarboximidamides 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.

Accuracy and precision of analytical method was also calculated
The accuracy of developed method was studied using single solutions at concentration levels ranging from 0.2 to 50 ng mL -1 and prepared mixtures containing also substrates of the reaction. The samples of known concentration were injected into the LC/MS/MS system. The peak area was used for calculating the N-alkyloxypyridinecarboximidamides concentrations using the corresponding regression equations (Tables 3, 4). Accuracy percentages were calculated. The method was found accurate for all studied compounds with average recovery of 97.72 ± 1.38, 100.81 ± 0.93, 99.03 ± 1.29, 96.99 ± 1.39, 98.89 ± 0.89 and 101.06 ± 1.12% for 2-Eh, 3-Eh, 4-Eh, 2-D, 3-D and 4-D, respectively. Precision was carried out by analyzing laboratory prepared mixtures of appropriate N-alkyloxypyridinecarboximidamide, alkyl bromide, N-hydroxypyridinecarboximidamide, NaOH, ethanol, water within the linearity range (Tables 3, 4) on the same day (n = 3) and on three consecutive days using the same procedure. The accuracy of the method for the selected concentrations was calculated using the corresponding regression equations and was found to be satisfactory. The percentage relative standard Table 3 Regression equations, linear ranges, LOD and LOQ parameters determined for N-(2-ethylhexyloxy)pyridine-2-carboximidamide (2-Eh), N-(2-ethylhexyloxy)pyridine-3-carboximidamide (3-Eh) and N-(2-ethylhexyloxy)pyridine-4-carboximidamide (4-Eh)

Analyte
Mass of degradation products deviation values for 2-Eh and 2-D were less than 1.5%, and for 3-Eh, 4-Eh, 3-D and 4-D were less than 1.0%.

Conclusions
The LC-MS/MS method to determine 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 was developed and validated in terms of linearity, precision and accuracy. The developed method is an accurate and easily performed method for determining amphiphilic N-alkyloxypyridinecarboximidamides directly from reaction mixture. The method was relatively unsusceptible to matrix effects, especially to the presence of reaction substrates. The limits of detections were at low levels (0.0125-0.05 ng mL -1 ) and precision was below 1.5%. The LC-MS/MS enables the identification, detection and quantitation of the N-alkyloxypyridinecarboximidamides at low concentration at real synthesis conditions. The LC-MS/MS N-alkyloxypyridinecarboximidamides determination at the real synthesis conditions enables monitoring of O-alkylation reaction progress. The data provided indicated that regardless on the alkyl bromide structure and position of aminoimineoxide moiety at pyridine ring, the intensive heating enabled a more efficient conversion of the substrates to the desired product.
The conducted studies (method validation and verification at real synthesis conditions) enable to select the MRM transition, which guarantee precise value of concentration of the synthesized N-alkyloxypyridinecarboximidamide.  Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.