Derivatization procedures for the detection of beta(2)-agonists by gas chromatographic/mass spectrometric analysis

An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives.     

Self-selection in olefin cross-metathesis: the effect of remote functionality

Olefin cross-metathesis (CM) is potentially an attractive method for generating dynamic combinatorial libraries (DCLs). In order for the CM reaction to be useful for DCL production, the course of the reaction and product distribution must be relatively insensitive to functionality remote from the reacting centers. We report on the CM of a series of allyl- and homoallylamides that are strongly dependent on remote functionality. This includes an unusual example of a cis-selective CM. [Reaction: see text]     

Micellar electrokinetic capillary chromatography for the determination of Viagra and its metabolite (UK-103,320) in human serum

Micellar electrokinetic capillary chromatography (MEKC) was investigated for the determination of Viagra (sildenafil citrate, SC) and its metabolite (UK-103,320) in human serum in a concentration range of clinical interest. For MEKC, human serum samples spiked with SC and UK were obtained directly after elution with methanol from a tC18 cartridge. The extract was evaporated and regenerated in a solution 1 mM of phosphate buffer (pH 12.3) which contained a methanol percentage of 20% that was analyzed using phosphate buffer (pH 12.3, 10 mM) containing 30 mM sodium dodecyl sulfate (SDS) as separation electrolyte and a fused-silica capillary. This method gave satisfactory interday precision with respect to migration times relative standard deviation (RSD < 1%) and linear responses for the concentration ranges investigated (0.50-3.50 mg L(-1) for the compound SC and 0.90-4.60 mg L(-1) for UK). An intraday RSD (n = 5 graphs) between the slopes of the calibration graphs was acceptable (6.40%) for SC and (3.37%) for UK. A satisfactory interday precision between slopes was also obtained (RSD 4.10% for SC and a RSD 2.72% for UK) which demonstrated the ruggedness of this method. Detection limits (S/N = 3) were about 200 ng/mL for both compounds in human serum. MEKC was shown as a good method with regards to simplicity, precision and sensitivity.     

A density functional theory study on the electrocyclization of 1,2,4,6-heptatetraene analogues: converting a pericyclic to a pseudopericyclic reaction

A comprehensive B3LYP/6-31+G* study on the electrocyclization of 1,2,4,6-heptatetraene analogues was conducted. Starting from the cyclization of (2Z)-2,4,5-hexatrienal, a pericyclic disrotatory process favored by the assistance of a electron lone pair, we incorporated small modifications in its molecular structure to obtain a truly pseudopericyclic process. To this purpose electronegative atoms (fluorine and nitrogen) were added to give a more electrophilic character on the carbon atom which is attacked by the electron lone pair of the oxygen atom. The complete pathway for each reaction was determined, and changes in magnetic properties were monitored with a view to estimating the aromatization associated with each process. This information, together with the energetic and structural results, allowed us to classify the reactions as pseudopericyclic or pericyclic. Among all studied reactions only one was a truly pseudopericyclic process and another was a borderline case. The features of this unequivocally pseudopericyclic case were analyzed in depth.     

MP2 study of substituent effects of 2-substituted alkyl ethyl methylcarbamates in homogeneous, unimolecular gas phase elimination reaction

Møller-Plesset MP2/6-31G method was used to examine the gas-phase elimination of 2-substituted alkyl ethyl N,N-dimethylcarbamates. The results of these calculations support a concerted non-synchronous six-membered cyclic transition state mechanism for carbamates containing a C_β–H bond at the alkyl side of the ester. These substrates produce the N,N-dimethylcarbamic acid and the corresponding olefin. The unstable intermediate, N,N-dimethylcarbamic acid, rapidly decomposes through a four-membered cyclic transition state to dimethylamine and CO₂ gas. Correlation of the logarithm of theoretical rate coefficients against original Taft's σ* values gave an approximate straight line (ρ*=−1.39, r=0.9558 at 360 °C). In addition to this fact, when log k_rel is plotted against the theoretical log k_rel for 2-substituted ethyl N,N-dimethylcarbamates a reasonable straight line (r=0.9919 at 360 °C) is obtained, suggesting similar mechanism.     

Synthesis of 4-aryl-2,3,6,7-tetrahydro-1H-pyrimido[4,5-b][1,4]-diazepin-6-ones from 4,5-diamino-1H-pyrimidin-6-ones and 1-aryl-3-(dimethylamino)-1-propanones

The reaction of 1-aryl-3-(dimethylamino)-1-propanones 1 with one equivalent of 4,5-diamino-1H-pyrimidin-6-ones 2 , in acidic medium, leads to the formation of 4-aryl-2,3,6,7-tetrahydro-1H-pyrimido[4,5-b]-[1,4]diazepin-6-ones 3 . The structure elucidation of the products is based on detail nmr analysis of experiments such as ¹³C, ¹H and DEPT including selective ¹³C{¹H} decoupling experiments.     

Semiclassical variational study of size effects in neutral and charged jellium droplets

An analytic expression for the total energy of metallic clusters composed ofN identical atoms of valencev and with net chargeQ is obtained by means of a variational solution of the Thomas-Fermi-Weizsäcker energy density functional within the spherical jellium model. The minimum energy is given as an expansion in decreasing powers of the cluster radiusR=r _s Z ^1/3, withZ=vN andr _s the radius per electron of the bulk metal. The coefficients are obtained as functions ofr _s . Terms of volume (R ³), surface (R ²), curvature (R), constant (R ⁰), (1/R) and (1/R ²) are clearly separated in the formula, as well as the different contributions (kinetic, coulombic and exchange-correlation) to each of them. The asymptotic values (R→∞) for the work functions,W(r _s ), and surface energies σ(r _s ), are compared to analogous semiclassical and Kohn-Sham calculations of jellium-like surfaces and to the experimental values. The size dependent behaviour of chemical potentials, μ(R), electron affinities,AF(R), and ionization potentials,IP(R), are easily obtained for any kind of metallic clusters. In particular we discuss the Coulomb and quantum corrections to the coefficients β, δ in the asymptotic formulae:IP?W+β/R andAF?W+δ/R.     

Synthesis and physicochemical study of the laminin active sequence: SIKVAV

The synthesis, physicochemical characterization, and interaction with membrane model systems of a peptide derived from the PA22-2 region of laminin are described. Surface activity studies indicate that this peptide is able to spread at the air-water interface being the maximal spreading pressure 20 mN/m at subphase concentrations around 10 micro M. Besides, these peptide molecules are also able to form stable monolayers. Physicochemical studies concerning the interaction of this peptide with lipids, organized in mono and bilayers, were carried out using Langmuir balance experiments and polarization fluorescence techniques. The peptide penetrates better in monolayers of DPPC than in those of PC and forms condensed mixed monolayers with DPPC. Energies of mixing are small thus indicating that deviations from ideality were almost negligible. Interactions with bilayers were studied through microviscosity changes (DPH and TMA-DPH probes), membrane permeability alterations (CF, NBD-PE/dithionite), and fusion promotion (NBD-PE/Rh-PE, resonance energy transfer). Results indicate that this sequence interacts very softly with bilayers without promoting changes in their organization. These data as well as the lack of interaction with erythrocytes suggest that coating liposomes with this peptide through chemical amide bonds can render stable inmunoliposomes for further biological applications.     

Terbium-sensitized luminescence determination of levofloxacin in tablets and human urine and serum

A selective and sensitive luminescence method for the determination of levofloxacin is described. The method is based in the luminescence signal from a terbium(III)-levofloxacin complex, in a micellar solution of sodium dodecyl sulfate (SDS), using a chemical deoxygenation agent (Na2SO3). The method allows the determination of 8-600 ng mL-1 of levofloxacin in 10 mM SDS solution containing 0.04 M acetic acid-sodium acetate buffer (pH 6) and 7.5 mM Na2SO3 with lambda exc = 292 nm and lambda em = 546 nm. The luminescence method was applied to the determination of the levofloxacin in a Spanish commercialized pharmaceutical formulation Tavanic (Hoechst Marion Roussel). Good concordance was found between the nominal and experimental values (500 and 488 mg, respectively), with a relative standard deviation (RSD) of 0.6%. The proposed method was shown to be 100-fold more sensitive than the spectrophotometric method, and nearly 2-fold more sensitive than the fluorescence method. The method was also applied to levofloxacin determination in human serum (by external calibration method) and urine (by standard additions method), spiked at levels found after drug administration at normal clinical doses. Average recoveries found were 90.1 (RSD 1%) and 102 (RSD 1.9%), respectively.