Preventive doping control screening analysis of prohibited substances in human urine using rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometry.

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, beta(2)-agonists, beta-blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single-step liquid-liquid extraction of hydrolyzed urine and the use of a rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4-methyl-2-hexanamine, which resulted in re-reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives.

Structural characteristics of anabolic androgenic steroids contributing to binding to the androgen receptor and to their anabolic and androgenic activities. Applied modifications in the steroidal structure.

Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone introduced for therapeutic purposes providing enhanced anabolic potency with reduced androgenic effects. Androgens mediate their action through their binding to the androgen receptor (AR) which is mainly expressed in androgen target tissues, such as the prostate, skeletal muscle, liver and central nervous system. This paper reviews some of the wide spectrum of testosterone and synthetic AAS structure modifications related to the intended enhancement in anabolic activity. The structural features of steroids necessary for effective binding to the AR and those which contribute to the stipulation of the androgenic and anabolic activities are also presented.

Fraction Lipophilicity Index (FLI). A drug-like metric for orally administered ionizable drugs.

Fraction Lipophilicity Index (FLI) was developed as a metric for assessing drug likeness of ionizable oral drugs. Considering that both log P and log D have distinct roles in drug action, the metric FLI allocates lipophilicity to a pH dependent neutral fraction of the molecule and is a weighted combination of log P and log D. It is expressed by equation: FLI = 2 x log P-│log D│. A dataset of 368 basic and acidic drugs was analyzed. Based on available % absorption data, drugs were classified into class 1 (268 drugs) and class 2 (100 drugs). The freeware MedChem Designer was used for log P and log D calculations at pH 7.4 and pH 5.5 for acids. Based on class 1, a drug-like FLI range 0-8 was defined. FLI distribution for class 2 is shifted towards significantly lower values. Comparison of FLI with rule of 5 (Ro5) shows that it leads to fewer values outside the established range than the corresponding log P violations for class 1. For class 2 it gives more alerts than Ro5 and can be considered complementary to Ro5, while it also sets lower limits to discriminate drugs with poor absorption.

Application of the ion pair concept to the n-octanol-water partitioning of cefepime and cefpirome.

The ion pair concept was applied for the assessment of lipophilicity of cefepime and cefpirome. Octanol-water distribution coefficients were determined in presence of different concentrations [X-] of sodium octanesulphonate. The log Dx values within the linear part of the log Dx/[X-] relationships were extrapolated to log Do values corresponding to the partitioning in absence of the counter ion. Measurements were feasible at pH values close to the isoelectric points of the acidic and basic functions. In that pH range the conduction of the experiments in presence of the hydrophobic counter anion facilitated the partitioning of the two cephalosporins to octanol, circumventing the problems arising from their high hydrophilicity. This procedure could not be applied at lower pH, possibly due to a further drastic decrease in the 'intrinsic' lipophilicity or to reduced ion pairing potential of octanesulphonate, and at higher pH due to the disruption of the zwitterionic structure. Extrapolated log Do values were compared to actual log D measurements performed for a reference quinolinium compound and for cefpirome. Extrapolated retention factors log kw close to the isoelectric point were also determined by reversed phase HPLC and compared to the log Do values.

Investigation of the retention/pH profile of zwitterionic fluoroquinolones in reversed-phase and ion-interaction high performance liquid chromatography.

The retention/pH profiles of three fluoroquinolones, ofloxacin, norfloxacin and ciprofloxacin, was investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC) and reversed-phase ion-interaction chromatography (RP-IIC), using an octadecylsilane stationary phase and acetonitrile as organic modifier. Sodium hexanesulphonate and tetrabutylammonium hydroxide were used as sources of counter ions in ion-interaction chromatography. The retention/pH profiles under in RP-HPLC were compared to the corresponding lipophilicity/pH profiles. Despite the rather hydrophilic nature of the three fluoroquinolones positive retention factors were obtained while there was a shift of the retention maximum towards more acidic pH values. This behavior was attributed mainly to non-hydrophobic silanophilic interactions with the silanized silica gel material of the stationary phase. In ion-interaction chromatography the effect of counter ions over a broad pH range was found to be ruled rather by the ion pair formation in the mobile phase which led to a drastic decrease in retention as a consequence of the disruption of the zwitterionic structure and thereupon the deliberation of a net charge in the molecules. At pH values at which zwitterionic structure was not favored both the ion-exchange and ion pair formation mechanisms were assumed to contribute to the retention.

Modelling of aldose reductase inhibitory activity of pyrrol-1-yl-acetic acid derivatives by means of multivariate statistics.

The inhibition of the aldose reductase enzyme (AR) is considered to be a promising approach to control chronic diabetes complications as well as a number of other pathological conditions. Thus considerable efforts are devoted to the development of aldose reductase inhibitors (ARIs) as possible pharmacotherapeutic agents. The establishment of adequate QSAR models would serve to this purpose. In the present study multivariate statistics was applied in order to analyse the AR inhibitory activity data of twenty three pyrrol-1-yl-acetic acid derivatives on the basis of essential molecular descriptors. The compounds contain one or two carbonyl keto groups, which serve as a bridge to link the pyrrole moiety to aromatic nuclei with or without further substitution. An adequate one component model with satisfactory statistics was obtained and validated for its robustness and predictive ability. The influence of the different descriptors in ARI activity is discussed. The derived model was further used to predict the activity of four independent compounds and the contribution of their specific structural characteristics in ARI activity was evaluated.

Prediction of distribution coefficient from structure. 2. Validation of Prolog D, an expert system.

Prolog D is a program that formalizes, in a controllable and reproducible manner, an algorithm developed to predict distribution coefficients of ionizable compounds at a given pH and varying counterion concentrations. Its predictive power has been evaluated with experimental log D values measured under standard conditions of buffers and ionic strength. Calculations were performed with the three different options for the estimation of partition coefficients (log P) implemented in the program. Considering the diversity of test compounds as well as the present state of the art in log P and pKa predictions, Prolog D proved to be very efficient and can be used as a tool to provide lipophilicity data. Prediction patterns and correlations with the observed data are of almost equal quality for all options, permitting acceptable results for 80% of the data.

Prediction of distribution coefficient from structure. 1. Estimation method.

A method has been developed for the estimation of the distribution coefficient (D), which considers the microspecies of a compound. D is calculated from the microscopic dissociation constants (microconstants), the partition coefficients of the microspecies, and the counterion concentration. A general equation for the calculation of D at a given pH is presented. The microconstants are calculated from the structure using Hammett and Taft equations. The partition coefficients of the ionic microspecies are predicted by empirical equations using the dissociation constants and the partition coefficient of the uncharged species, which are estimated from the structure by a Linear Free Energy Relationship method. The algorithm is implemented in a program module called PrologD.

Kinetics of the acidic and enzymatic hydrolysis of benazepril HCl studied by LC.

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed and validated for the kinetic investigation of the chemical and enzymatic hydrolysis of benazepril hydrochloride. Kinetic studies on the acidic hydrolysis of benazepril hydrochloride were carried out in 0.1 M hydrochloric acid solution at 50, 53, 58 and 63 degrees C. Benazepril hydrochloride appeared stable in a pH 7.4 phosphate buffered solution at 37 degrees C and showed susceptibility to undergoing in vitro enzymatic hydrolysis with porcine liver esterase (PLE) in a pH 7.4 buffered solution at 37 degrees C. Benazeprilat appeared to be the major degradation product in both (chemical and enzymatic) studies of hydrolysis. Statistical evaluation of the proposed HPLC methods revealed their good linearity and reproducibility. Relative standard deviation (R.S.D.) was less than 4.76, while detection limits for benazepril hydrochloride and benazeprilat were 13.0 x 10(-7) and 9.0 x 10(-7) M, respectively. Treatment of the kinetic data of the acidic hydrolysis was carried out by non-linear regression analysis and k values were determined. The kinetic parameters of the enzymatic hydrolysis were determined by non-linear regression analysis of the data using the equation of Michaelis-Menten.

Synthesis, lipophilicity and biological evaluation of indole-containing derivatives of 1,3,4-thiadiazole and 1,2, 4-triazole.

3-[(2-Methyl-1H-3-indolyl) methyl]-4-aryl-4, 5-dihydro-1H-1,2,4-triazole-5-thiones 6a-c and their respective N-¿5-[2-methyl-1H-3-indolyl) methyl]-1,3,4-thiadiazol-2-yl¿-N-arylamines 7a,b have been prepared. The antidepressant profile of 6a,c and 7a was studied on mice with respect to that of the analogous 3-(1H-1-indolylmethyl)-4-aryl-4,5-dihydro-1H-1,2,4-triazole-5-thio nes 1a-c and the respective N-¿5-[(2-methyl-1H-3-indolyl) methyl]-1,3,4-thiadiazole-2-yl¿-N-arylamines 2a-c, the synthesis and antimicrobial potency of which we have recently reported. Behavioral effects, induced by the members of both series, in conjunction with their activity in some specific tests (forced swim, pentetrazole convulsions) on mice, show that these derivatives cross the blood-brain barrier and could develop an antidepressant activity comparable to that of imipramine. Blood-brain barrier penetration is also supported by the lipophilicity data obtained for all analogs.

Synthesis, lipophilicity and biological properties of some novel 1H-1,2,4 triazole derivatives.

A series of new 1H-1,2,4-triazole derivatives was synthesized and evaluated as potential antiviral (i.e. anti-influenza virus), antibacterial and antifungal agents. The lipophilicity of the compounds was also investigated using calculation procedures. Among the test compounds none showed specific activity against influenza virus, although compound 3a, the most hydrophilic member of the series, showed weak activity against Bacillus subtillis.

Synthesis, lipophilicity and antimicrobial properties of some O-(5-aryl-1,2,4-triazol-3-yl-ethyl)benzaldoximes and O-(5-aryl-1,3,4-oxydiazol-2-yl-ethyl)benzaldoximes.

The synthesis of benzaldehyde oximethers with suitably substituted 1,2,4-triazoles and 1,3,4-oxadiazoles from the corresponding benzaldoximes is described. The lipophilicity of the compounds was measured as well as their antimicrobial and antifungal activity in vitro. Certain compounds showed relatively significant activity against Bacillus subtilis.

Prediction of distribution coefficients from structure. Comparison of calculated and experimental data for various drugs.

The efficiency of the program PrologD to predict distribution coefficients (D) at any pH and pairing ion concentration has been tested using experimental logD values for various drugs measured under standard conditions of buffers and ionic strength. Clonidine derivatives, fluoroquinolones and beta-blockers were included as particular pharmacological classes within the testing data set. Calculations were performed using the three logP estimation options implemented in the program. PrologD proved to be very efficient and can be of great advantage in drug research. Prediction patterns and correlations between experimental and calculated data indicate acceptable results for more than 80% of the data. In addition, comparable studies using the different options permitted suggestions for the more suitable logP estimation method in respect of the particular classes of compounds.

Comparison of multiple linear regression, partial least squares and artificial neural networks for prediction of gas chromatographic relative retention times of trimethylsilylated anabolic androgenic steroids.

The comparison among different modelling techniques, such as multiple linear regression, partial least squares and artificial neural networks, has been performed in order to construct and evaluate models for prediction of gas chromatographic relative retention times of trimethylsilylated anabolic androgenic steroids. The performance of the quantitative structure-retention relationship study, using the multiple linear regression and partial least squares techniques, has been previously conducted. In the present study, artificial neural networks models were constructed and used for the prediction of relative retention times of anabolic androgenic steroids, while their efficiency is compared with that of the models derived from the multiple linear regression and partial least squares techniques. For overall ranking of the models, a novel procedure [Trends Anal. Chem. 29 (2010) 101-109] based on sum of ranking differences was applied, which permits the best model to be selected. The suggested models are considered useful for the estimation of relative retention times of designer steroids for which no analytical data are available.

Gas chromatographic quantitative structure-retention relationships of trimethylsilylated anabolic androgenic steroids by multiple linear regression and partial least squares.

A quantitative structure-retention relationship (QSRR) study has been performed to correlate relative retention times (RRTs) of trimethylsilylated (TMS) anabolic androgenic steroids (AAS) with their molecular characteristics, encoded by the respective descriptors, for the prediction of RRTs of novel molecules, using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). The elucidation of similarities and dissimilarities among the data structures was carried out using principal component analysis (PCA). Successful models were established using multiple linear regression (MLR) and partial least squares (PLS) techniques as a function of topological, three-dimensional (3D) and physicochemical descriptors. The models are useful for the estimation of RRTs of designer steroids for which no analytical data is available.

Schemes of metabolic patterns of anabolic androgenic steroids for the estimation of metabolites of designer steroids in human urine.

Unified metabolism schemes of anabolic androgenic steroids (AAS) in human urine based on structure classification of parent molecules are presented in this paper. Principal components analysis (PCA) was applied to AAS molecules referred in the World Anti-Doping Agency (WADA) list of prohibited substances, resulting to their classification into six distinct groups related to structure features where metabolic alterations usually occur. The metabolites of the steroids participating to these six groups were treated using the Excel(c) classification filters showing that common metabolism routes are derived for each of the above PCA classes, leading to the proposed metabolism schemes of the present study. This rule-based approach is proposed for the prediction of the metabolism of unknown, chemically modified steroids, otherwise named as designer steroids. The metabolites of three known, in the literature, AAS are estimated using the proposed metabolism schemes, confirming that their use could be a useful tool for the prediction of metabolic pathways of unknown AAS.

A quantitative structure-activity relationship (QSAR) study of alkylpyrazine odor modalities.

The odor strength of a series of alkylpyrazines has been quantitatively investigated applying molecular connectivity, molecular shape, and the recently developed electrotopological state indices. The successful use of the latter parameters indicates that both electronic and topological features contribute to the odor strength of the compounds under study, while the specific role of the two nitrogen atoms is revealed.

Synthesis, lipophilicity studies and antibacterial properties of some novel quaternary ammonium salts.

The synthesis of some novel quaternary ammonium salts, derivatives of 15-phenyl-decapentanoic acid, is described. Their lipophilicity was estimated applying the Hansch-Leo fragmental procedure and measured by means of reversed phase thin layer chromatography. All compounds were tested for their antibacterial activity against Gram positive and gram negative microorganisms. The less lipophilic compounds showed weak activity, mainly against gram positive microorganisms.

Synthesis and biological evaluation of indole containing derivatives of thiosemicarbazide and their cyclic 1,2,4-triazole and 1,3,4-thiadiazole analogs.

New indolic derivatives of thiosemicarbazides and some cyclic 1,2,4-triazol-5-thione analogs were synthesized. The newly synthesized compounds as well as some indole containing thiosemicarbazides, 1,2,4-triazoles and 1,3,4- thiadiazoles, which have been reported previously, were investigated for antimicrobial, antifungal and antiphage activity. Certain thiosemicarbazide derivatives and the corresponding cyclic 1,2,4-triazole analogs showed selective antimicrobial or antifungal activity, while they lack any antiphage activity. Antiphage activity was detected for one compound, bearing the 1,3,4-thiadiazole nucleus. The selectively active compounds cover a wide range of lipophilicity. Structure-activity relations show a remarkably similarity in the antimicrobial and antifungal behaviour of the thiosemicarbazides and their cyclic triazo-thien-5-yl analogs, while alpha-naphtyl substitution in the non indolic portion of the molecule is favorable. C5 substitution on the indolic nucleus may also be critical for selective activity.

Ligand specificity of the genetic variants of human alpha1-acid glycoprotein: generation of a three-dimensional quantitative structure-activity relationship model for drug binding to the A variant.

Human alpha1-acid glycoprotein (AAG) is a mixture of at least two genetic variants: the A variant and the F1 and/or S variant or variants, which are encoded by two different genes. In a continuation of previous studies indicating specific drug transport roles for each AAG variant according to its separate genetic origin, this work was designed to (1) determine the affinities of the two main gene products of AAG (i.e., the A variant and a mixture of the F1 and S variants) for 35 chemically diverse drugs and (2) to obtain meaningful 3D-QSARs for each binding site. Affinities were obtained by displacement experiments, leading to qualitative indications about binding site characteristics. In particular, drugs binding selectively to the A variant displayed some common structural features, but this was not seen for the F1*S variants. Three-dimensional QSAR analyses using the CoMFA method yielded a steric model for binding to the A variant, from which a simplified haptophoric model was derived. In contrast, no statistically sound model was found for the F1*S variants, possibly due (among other reasons) to an insufficient number of high affinity ligands in the set.