Validation of Analytical Methods for Tacrolimus Determination in Poly(ε-caprolactone) Nanocapsules and Identification of Drug Degradation Products.

The aim of this paper was to use chromatographic tools for validating an analytical method for the tacrolimus (TAC) determination in polymeric nanocapsules and for identifying the drug degradation products after alkaline stress. A rapid Ultra-High-Performance Liquid Chromatography coupled with photo-diode array (UHPLC-PDA) method was successfully performed using the following chromatographic conditions: the Shimadzu Shim-pack XR-ODS III C18 column (100 mm×2.00 mm, 2.2 µm), the mobile phase consisting of methanol and acidified ultrapure water (89:11 v/v), the flow rate of 0.55 mL·min-1, and the ultraviolet (UV) detection at 235 nm. This method was validated as per International Council for Harmonisation (ICH) guidelines. In addition, a TAC forced degradation assay was carried out after alkaline stress and its degradation products were investigated using Liquid Chromatography coupled tandem mass spectroscopy (LC-MS/MS). The calibration curve was linear in the range of 100.0-300.0 µg·mL-1 (r >0.9999). Accuracy was confirmed by the TAC recovery of 96.55 to 98.19%. Precision (intraday and interday) were demonstrated by relative standard deviation lower than 0.89% and 3.25%, respectively. Selectivity and robustness were also proved. The method developed it was successfully applied to quantify TAC from polymeric nanocapsules, showing a high loading efficiency rate (>96.47%). The main drug degradation product observed in a multiple reaction monitoring (MRM) experiment was m/z 844, confirming the susceptibility of TAC under alkaline conditions; this finding was first time described.

Theoretical and experimental investigation on the rotational isomerism in alpha-fluoroacetophenones.

The geometries involved in the conformational equilibria of alpha-fluoroacetophenone, p-nitro-alpha-fluoroacetophenone, and p-methoxy-alpha-fluoroacetophenone were investigated. Theoretical calculations showed that cis and gauche forms (F-C-C=O) are their most stable conformers and that in the vapor phase the gauche conformer is predominant. The three compounds were synthesized, and the conformational behavior in solution was estimated from infrared (IR) and nuclear magnetic resonance (NMR) spectra obtained in solvents of different polarity. Their IR spectra showed one carbonyl absorption for the cis and one for the gauche conformer, and that the cis conformer was preferred in the more polar solvents. (1)J(CF), (2)J(C(O)F), and (2)J(HF) coupling constants were obtained from their NMR spectra, and they also showed a preference for the cis conformer when more polar solvents were used. The vapor phase calculations showed a conformational preference for the gauche form. However, when the solvent effects were included in the calculations, the results were in complete agreement with the experimental data (NMR and IR), the cis conformer being the most stable one.

Conformational equilibrium of phenylacetic acid and its halogenated analogues through theoretical studies, NMR and IR spectroscopy.

This paper presents a study on the conformational preferences of phenylacetic acid (PA) and its halogenated analogues (FPA, CPA, BPA). To clarify the effects that rule these molecules' behaviour, theoretical calculations were used, for both the isolated phase and solution, combined with nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. Most conformations of phenylacetic acid and its halogenated derivatives are stabilized through the hyperconjugative effect, which rules the conformational preference. NMR analyses showed that even with the variation in medium polarity, there was no significant change in the conformation population. Infrared spectroscopy showed similar results for all compounds under study. In most spectra, two bands were found through the carbonyl deconvolution, which is in accordance with the theoretical data. It was possible to prove that variation in the nature of the substituent in the ortho position had no significant influence on the conformational equilibrium.

Rotational isomerism of some chloroacetamides: theoretical and experimental studies through calculations, infrared and NMR.

The geometries involved in the conformational equilibria of 2,2-dichloro-N-cyclohexyl-N-methyl-acetamide (DCCMA) and 2-chloro-N,N-dicyclohexylacetamide (CDCA) were investigated. Theoretical calculations at the B3LYP/cc-pVDZ level of theory showed that gauche forms (ClCCO) are the most stable and the predominant conformers in isolated phase. Both compounds had the conformational behavior in solvents of different polarities estimated from theoretical calculations with the PCM (Polarizable Continuum Model), at the same level of theory, using infrared data from deconvolution of the carbonyl absorption bands and (13)C NMR spectra. Their IR spectra showed two carbonyl absorptions and that the conformer with the highest dipole moment had its population increased when the most polar solvents were used, in accordance with the theoretical calculation in solution. (1)JCH coupling constants were obtained from their NMR spectra, and revealed that there was population variation of conformers with solvent exchange. Experimental data (NMR and IR) as well as calculations including the solvent effects followed the same trend.

Studies on the s-cis-trans isomerism for some furan derivatives through IR and NMR spectroscopies and theoretical calculations.

The s-cis-trans isomerism of two furan derivatives [2-acetyl- (AF) and 2-acetyl-5-methylfuran, (AMF)] was analyzed, using data from the deconvolution of their carbonyl absorption band in two solvents (CH(2)Cl(2) and CH(3)CN). These infrared data showed that the O,O-trans conformers predominate in the less polar solvent (CH(2)Cl(2)), but these equilibria change in a more polar solvent (CH(3)CN) leading to a slight predominance of the O,O-cis conformers, in agreement with the theoretical calculations. The later results were obtained using B3LYP-IEFPCM/6-31++g(3df,3p) level of theory, which taking into account the solvent effects at IEFPCM (Integral Equation Formalism Polarizable Continuum Model). Low temperature (13)CNMR spectra in CD(2)Cl(2) (ca. -75 °C) showed pairs of signals for each carbon, due to the known high energy barrier for the cis-trans interconversion leading to a large predominance of the trans isomers, which decreases in acetone-d(6). This was confirmed by their (1)HNMR spectra at the same temperatures. Moreover, despite the larger hyperconjugative interactions for the O,O-cis isomers, obtained from NBO data, these isomers are destabilized by the their Lewis energy.

Conformational preferences for some 5-substituted 2-acetylthiophenes through infrared spectroscopy and theoretical calculations.

The s-cis-trans isomerisms of some derivatives of thiophene (2-acetyl, AT; 2-acetyl-5-bromo, ABT and 2-acetyl-5-chloro, ACT) were analyzed, using data from deconvolution of their carbonyl absorption bands in two solvents (CCl4 and CHCl3). These infrared data showed that the O,S-cis conformer largely predominates in the studied solvents and that the same occurs in the gas phase, as observed from theoretical calculations. The latter results were obtained using B3LYP/6-311++G(3df,3p) and MP2/6-311++G(3df,3p) levels of theory, with zero-point energy correction. Moreover, the use of the IEFPCM (Integral Equation Formalism Polarizable Continuum Model) to take into account the solvent effects, using the same levels of theory, confirmed the results observed from infrared data. Low temperature 13C NMR spectra in CS2/CD2Cl2 (-90 °C) and in acetone-d6 (-80°C) did not show pairs of signals for each carbon, due to the known low energy barrier (∼8 kcal mol(-1)) for the cis-trans interconversion. Data from NBO calculations show that the nO(2)→σS-C5* and nO(2)→σC2-C3* interactions occur only in the O,S-cis isomer and can explain its conformational preference.

Conformational behavior of cis-2-methoxy, cis-2-methylthio, and cis-2-methylselenocyclohexanol: a theoretical and experimental investigation.

Studies on the conformational equilibria of 2-methoxy, 2-methylthio, and 2-methylselenocyclohexanol are reported. Dynamic NMR spectroscopy experiments at 203-210 K were performed, which provided the percentages of each conformer in equilibrium. Theoretical calculations using the B3LYP method and aug-cc-pvdz basis set were applied to determine the differences in energy between the conformers. The analysis of the potential energy surface of each conformer showed the presence of two rotamers. Natural bond orbital analysis provided an explanation of which factors are driving the rotamer and conformer preferences.