Fully automated electrophoretically mediated microanalysis for CYP1A1 activity monitoring optimized by multivariate approach.

In this study, a fully automated incapillary system was developed to monitor the activity of CYP1A1 (Cytochrome P450, family 1, subfamily A, polypeptide 1) in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an inline bioreaction in the presence of CYP1A1 supersomes and Nicotinamide adenine dinucleotide phosphate reduced as cofactor, giving rise to hydroxycoumarin, the product that was assayed. The optimization of the experimental conditions was supported by the application of a design of experiment, providing a better understanding of electrophoretic mixing parameters that influence the metabolic reactions. The results obtained in optimal conditions were compared not only to those achieved after offline metabolization but also with liver microsomes. Finally, inhibition studies were conducted showing an important decrease of hydroxycoumarin formation using apigenin as CYP1A1 potent inhibitor. This study demonstrates the usefulness of our inline system for the fully automated in vitro metabolism studies and the screening of new CYP1A1 inhibitors.

An Intravenous Pharmacokinetic Study of Cannabidiol Solutions in Piglets through the Application of a Validated Ultra-High-Pressure Liquid Chromatography Coupled to Tandem Mass Spectrometry Method for the Simultaneous Quantification of CBD and Its Carboxylated Metabolite in Plasma.

Cannabidiol (CBD) has multiple therapeutic benefits that need to be maximized by optimizing its bioavailability. Numerous formulations are therefore being developed and their pharmacokinetics need to be studied, requiring analytical methods and data from intravenous administration. As CBD is susceptible to hepatic metabolism, the requirement of any method is to quantify metabolites such as 7-COOH-CBD. We demonstrated that CBD and 7-COOH-CBD could be simultaneously and correctly quantified in piglet plasma by using an UHPLC-MS/MS technique. The validated method allowed for an accurate bioanalysis of an intravenously injected solution consisting of CBD-HPßCD complexes. The experimental pharmacokinetic profile of CBD showed multi-exponential decay characterized by a fast apparent distribution half-life (0.25 h) and an elimination half-life of two hours. The profile of 7-COOH-CBD was not linked with the first-pass metabolism, since 80% of the maximum metabolite concentration was reached at the first sampling time point, without any decrease during the period of study. A two-compartment model was optimal to describe the experimental CBD profile. This model allowed us to calculate macro-micro constants and volumes of distribution (Vss = 3260.35 ± 2286.66 mL) and clearance (1514.5 ± 261.16 mL·h-1), showing that CBD is rapidly distributed to peripheral tissues once injected and slowly released into the bloodstream.

Triphenylphosphonium salts of 1,2,4-benzothiadiazine 1,1-dioxides related to diazoxide targeting mitochondrial ATP-sensitive potassium channels.

The present work aims at identifying new ion channel modulators able to target mitochondrial ATP-sensitive potassium channels (mitoKATP channels). An innovative approach should consist in fixing a cationic and hydrophobic triphenylphosphonium fragment on the structure of known KATP channel openers. Such phosphonium salts are expected to cross the biological membranes and to accumulate into mitochondria. Previous works revealed that the presence of an (R)-1-hydroxy-2-propylamino chain at the 3-position of 4H-1,2,4-benzothiadiazine 1,1-dioxides KATP channel openers increased, in most cases, the selectivity towards the pancreatic-type (SUR1/Kir6.2) KATP channel. In order to target cardiac mitoKATP channels, we decided to introduce a triphenylphosphonium group through an ester link on the SUR1-selective (R)-7-chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide. The new compounds were found to preserve an inhibitory activity on insulin secretion (SUR1-type KATP channel openers) while no clear demonstration of an impact on mitochondria from cardiomyocytes (measurement of oxygen consumption, respiratory parameters and ATP production on H9C2 cells) was observed. However, the most active (inhibition of insulin release) compound 17 was found to penetrate the cardiac cells and to reach mitochondria.

Evaluation of chlorine containing cellulose-based chiral stationary phases for the LC enantioseparation of basic pharmaceuticals using polar non-aqueous mobile phases.

The discrimination ability of three cellulose-based chiral stationary phases (CSPs) was evaluated towards the enantiomers of basic drugs, using ACN as the main solvent in polar organic mobile phases. The study was focused on CSPs containing cellulose tris(3-chloro-4-methylphenylcarbamate) (3-Cl-4-MePC), cellulose tris(4-chloro-3-methylphenylcarbamate) (4-Cl-3-MePC) or cellulose tris(3,5-dichlorophenylcarbamate) (3,5-diClPC) as the chiral selector. The behaviour of these CSPs was studied systematically in order to investigate the influence of the presence and position of the chlorine substituents on the phenylcarbamate moieties on the retention and resolution of the enantiomers. The evaluation was made with three different generic mobile phases, namely ACN/0.1%DEA/0.1% TFA (DEA, diethylamine), ACN/0.1%DEA/0.2% FA and ACN/0.1%DEA/0.2%AcA, deduced from the previous study. The nature of the acidic additive and of the chiral selector was found to be particularly important for the retention and enantioresolution of these basic compounds. High-resolution values could be obtained for most studied enantiomers with these CSPs, clearly demonstrating the interest of using them in combination with polar organic mobile phases. However, significant differences in enantioresolution between the CSPs have been observed for many compounds, indicating that these phases seem to be quite complementary.

Optimization of micro-HPLC peak focusing for the detection and quantification of low hepcidin concentrations.

Micro-high-performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 µL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile-phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated.

Determination of enantiomeric purity of S-amlodipine by chiral LC with emphasis on reversal of enantiomer elution order.

An LC method was developed and prevalidated for the enantiomeric purity determination of S-amlodipine in polar organic solvent chromatography using a chlorine-containing cellulose-based chiral stationary phase (CSP). The concentration of formic acid (FA) (0.01-0.2%) in the mobile phase containing acetonitrile as the main solvent was found to influence the elution order of amlodipine enantiomers as well as the enantioresolution. A reversal of the enantiomer elution order of amlodipine was only observed with chiral stationary phases with both electron-withdrawing (chloro) and electron-donating groups (methyl) on the phenyl moieties of the chiral selector, namely cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(4-chloro-3-methylphenylcarbamate). The highest enantioresolution (Rs : 4.1) value was obtained at the lowest FA concentration (0.01%) using cellulose tris(4-chloro-3-methylphenylcarbamate) as the chiral selector and the enantiomeric impurity, R-amlodipine, eluted first under these conditions. Therefore, the mobile phase selected for the prevalidation of the method consisted of ACN/0.1% DEA/0.01% FA and the temperature was set at 25°C. The method was prevalidated by means of the strategy based on the total measurement error and the accuracy profile. The method was found to be selective and the limit of quantification was found to be about 0.05% for R-amlodipine, while the limit of detection was close to 0.02%.

Coupling of liquid chromatography/tandem mass spectrometry and liquid chromatography/solid-phase extraction/NMR techniques for the structural identification of metabolites following in vitro biotransformation of SUR1-selective ATP-sensitive potassium channel openers.

SUR1-selective ATP-sensitive potassium channel openers (PCOs) have been shown to be of clinical value for the treatment of several metabolic disorders, including type I and type II diabetes, obesity, and hyperinsulinemia. Taking into account these promising therapeutic benefits, different series of 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides structurally related to diazoxide were developed. In view of the lead optimization process of the series, knowledge of absorption, distribution, metabolism, excretion, and toxicity parameters, and more particularly the metabolic fate of these compounds, is a fundamental requirement. For such a purpose, two selected promising compounds [7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 73) and 7-chloro-3-(3-pentylamino)-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 157)] were incubated in the presence of phenobarbital-induced rat liver microsomes to produce expected mammal in vivo phase I metabolites. The resulting major metabolites were then analyzed by both mass spectrometry (MS) and NMR to completely elucidate their chemical structures. The two compounds were also further incubated in the presence of nontreated rats and human microsomes to compare the metabolic profiles. In the present study, the combined use of an exact mass liquid chromatography (LC)/tandem MS platform and an LC/solid-phase extraction/NMR system allowed the clarification of some unresolved structural assessments in the accurate chemical structure elucidation process of the selected PCO drugs. These results greatly help the optimization of the lead compounds.

Association of two single-isomer anionic CD in NACE for the chiral and achiral separation of fenbendazole, its sulphoxide and sulphone metabolites: application to their determination after in vitro metabolism.

A NACE method was developed for the separation of fenbendazole (FBZ), a prochiral drug giving rise to chiral (oxfendazole or OFZ) and nonchiral (FBZ sulphone or FBZSO(2)) metabolites. First, the effect of the nature and the concentration of CD as well as that of the acidic BGE on the enantiomeric separation of OFZ were studied. OFZ enantiomers were completely resolved using a BGE made up of 10 mM ammonium formate and 0.5 M TFA in methanol containing 10 mM heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-CD and 10 mM heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD. Moreover, the NACE method was found to be particularly well suited to the simultaneous determination of FBZ, OFZ enantiomers, and FBZSO(2). Thiabendazole was selected as an internal standard. The CD-NACE potential was then evaluated for in vitro metabolism studies using FBZ as a model case. The OFZ enantiomers and FBZSO(2) could be detected after incubation of FBZ in the phenobarbital-induced male rat liver microsomes systems.

Optimization of the LC enantioseparation of chiral pharmaceuticals using cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral selector and polar non-aqueous mobile phases.

The resolving power of a new commercial polysaccharide-based chiral stationary phase, Sepapak-4, with cellulose tris(4-chloro-3-methylphenylcarbamate) coated on silica microparticles as chiral selector, was evaluated toward the enantioseparation of ten basic drugs with widely different structures and hydrophobic properties, using ACN as the main component of the mobile phase. A multivariate approach (experimental design) was used to screen the factors (temperature, n-hexane content, acidic and basic additives) likely to influence enantioresolution. Then, the optimization was performed using a face-centered central composite design. Complete enantioseparation could be obtained for almost all tested chiral compounds, demonstrating the high chiral discrimination ability of this chiral stationary phase using polar organic mobile phases made up of ACN and containing an acidic additive (TFA or formic acid), 0.1% diethylamine and n-hexane. These results clearly illustrate the key role of the nature of the acidic additive in the mobile phase.

Effect of the nature of the single-isomer anionic CD and the BGE composition on the enantiomeric separation of beta-blockers in NACE.

The separation of ten beta-blockers has been investigated in NACE systems using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD (HDMS-beta-CD) and heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-CD (HDAS-beta-CD). The influence on enantioresolution, mobility difference and selectivity of the nature of both anionic CD and BGE anion as well as their concentrations were studied by means of a multivariate approach. A D-optimal design with 25 experimental points was applied. For all studied analytes, the enantiomeric resolution was shown to be significantly influenced by both CD nature and concentration. Except for two compounds, HDAS-beta-CD was found to give higher enantioresolution values than HDMS-beta-CD. The best enantioseparation for all compounds was achieved in the presence of a high chiral selector concentration and for most of them at a low BGE anion concentration. For each investigated compound, operating conditions leading to the best enantiomeric resolution were deduced. A generic NACE system was then recommended, namely 10 mM ammonium acetate and 40 mM HDAS-beta-CD in methanol acidified with 0.75 M formic acid. This generic system was able to completely resolve the enantiomers of all beta-blockers, with a R(s) value of at least 4. Finally, the optimal conditions obtained modelling resolution, mobility difference and selectivity were compared.

Nonaqueous capillary electrophoretic behavior of 2-aryl propionic acids in the presence of an achiral ionic liquid. A chemometric approach.

Ionic liquids (ILs) appear really attractive as electrolyte additives in nonaqueous capillary electrophoresis (NACE). These salts may offer new possibilities of interactions to modulate analyte effective mobilities. The presence of 1-n-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (BMIM NTf2) in acetonitrile/alcohol background electrolytes (BGEs) was investigated in this work. The aim of this study was to elucidate the influence of the IL concentration on the electrophoretic behavior of four arylpropionic acids and to identify the interactions between the analytes and the IL cation. The influence on mobility of the IL concentration, the nature and the proportion of the organic solvents, and the concentration of the ionic components of the BGE was first studied by a univariate approach. A four-factor D-optimal experimental design was then applied to provide a deeper insight into analyte interaction with IL cation present both free in BGE and adsorbed onto the capillary wall.

Development of a mass spectrometry method for the determination of a melanoma biomarker, 5-S-cysteinyldopa, in human plasma using solid phase extraction for sample clean-up.

5-S-cysteinyldopa is a well-known pigment intermediate and analysis of its plasma concentration is interesting for the early diagnosis, as well as for evaluation of treatment and follow-up of malignant melanoma. A determination method of 5-SCD in human plasma was developed using solid phase extraction (SPE) on disposable cartridges and liquid chromatography electrospray mass spectrometry (LC-ESI-MS-MS). Compound's sensitivity to light and oxidation requires the addition of anti-oxidative agents (AO), to work in acidic media at 4 degrees C and to avoid light exposure of samples since blood collection. Different solid phases involving covalent binding to phenylboronic groups or dual retention mechanisms were evaluated and extraction cartridges containing both hydrophobic and strong cation exchange functionalities were finally selected. The LC separation of 5-SCD from endogenous catecholamines was achieved by gradient elution on a C18 stationary phase. 5-SCD was detected by multiple reaction monitoring (MRM) performed on ES(+) generated ions. Finally, the method was prevalidated in the lower ng/ml range. Good results with respect to accuracy, trueness and precision were obtained.

Nonaqueous capillary electrophoresis method for the enantiomeric purity determination of S-timolol using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin: validation using the accuracy profile strategy and estimation of uncertainty.

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the enantiomeric purity determination of S-timolol maleate using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin (HDMS-beta-CD) as chiral selector. With a background electrolyte made up of a methanolic solution of 0.75 M formic acid, 30 mM potassium camphorsulfonate and containing 30 mM HDMS-beta-CD, the determination of 0.1% of R-timolol in S-timolol could be performed with an enantiomeric resolution of 8.5. Pyridoxine was selected as internal standard. The NACE method was then fully validated by applying a novel strategy using accuracy profiles. It is based on beta-expectation tolerance intervals for the total measurement error which includes trueness and intermediate precision. The uncertainty of measurements derived from beta-expectation tolerance intervals was estimated at each concentration level of the validation standards. To confirm the suitability of the developed and validated method, several real samples of S-timolol maleate containing R-timolol maleate at different concentrations were analysed and the results were compared to those obtained by liquid chromatography.

Pharmacokinetic study of a new synthetic MMP inhibitor (Ro 28-2653) after IV and oral administration of cyclodextrin solutions.

Ro 28-2653 (5-biphenyl-4-yl-5-[4-(4-nitro-phenyl)-piperazin-1-yl]-pyrimidine-2,4,6-trione) is a new synthetic inhibitor of matrix metalloproteinases (MMPs) with a high selectivity towards MMP2, MMP9 and membrane type 1-MMP. It has been shown that cyclodextrins (CDs) are able to form inclusion complexes with Ro 28-2653 and to increase its aqueous solubility. The aim of this study is to demonstrate that an increase in Ro 28-2653 solubility, via ternary complex formation, can lead to an increase in the oral bioavailability of this drug. This study shows that a synergistic effect exists between hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and l-lysine. The use of this multicomponent system enabled the preparation of oral and intravenous solutions of Ro 28-2653. In vivo evaluation of the oral solution of the inclusion complex of Ro 28-2653 in comparison with a suspension of the same uncomplexed drug showed a significant (p<0.05) increase in absolute bioavailability. The area under curve (AUC) and the peak serum concentration (Cmax) were approximately 10 times higher than those obtained with the suspension, while the time (Tmax) to reach Cmax was reduced. Moreover, in vivo administration of Ro 28-2653 solutions highlighted some information about the pharmacokinetic behavior of Ro 28-2653: a long biologic half-life (about 15.5h) and a small overall volume of distribution (8l).

Influence of the nature of the electrolyte on the chiral separation of basic compounds in nonaqueous capillary electrophoresis using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin.

The influence on the enantiomeric resolution of the nature of the cationic BGE component (sodium, ammonium or potassium) and that of the anionic component (chloride, formate, methanesulfonate or camphorsulfonate) as well as the concentration of heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin (HDMS-beta-CD), the selected chiral selector, was studied in nonaqueous capillary electrophoresis (NACE). For this purpose, two D-optimal designs with 33 and 26 experimental points were applied. Three beta-blockers (atenolol, celiprolol and propranolol) and three local anesthetics (bupivacaine, mepivacaine and prilocaine) were selected as basic model compounds. Both cationic and anionic BGE components were found to have a deep impact on the enantiomeric resolution of the investigated analytes but it is the cationic component that has shown the strongest influence. Indeed, in some cases, the change of the latter led to a complete loss of enantioresolution. Based on the observed results, two NACE systems were recommended, namely ammonium formate and potassium camphorsulfonate in a methanolic solution containing HDMS-beta-CD and acidified with formic acid, in order to separate efficiently the enantiomers of basic drugs.

Development and validation of a high-performance liquid chromatographic method for the determination of cyproterone acetate in human skin.

In the framework of a preliminary study on the transdermal penetration of cyproterone acetate (CPA), a simple and rapid procedure involving an extraction step coupled to a HPLC-UV determination has been developed for the separation and quantification of CPA in the two main skin layers-epidermis and dermis-after local application. The separation of epidermis and dermis layers was carefully carried out by means of a sharp spatula after skin immersion in heated water at 65 degrees C. The two skin layers were then treated separately according to the same process: (1) sample homogenization by vibration after freezing with liquid nitrogen in a Mikro-Dismembrator; (2) CPA extraction with methanol after addition of the internal standard (betamethasone dipropionate); (3) centrifugation; (4) evaporation of a supernatant aliquot; (5) dissolution of the dry residue in methanol and addition of water; (6) centrifugation; (7) injection of a supernatant aliquot into the HPLC system. The separation was achieved on octadecylsilica stationary phase using a mobile phase consisting in a mixture of acetonitrile and water (40:60 (v/v)). The method was then validated using a new approach based on accuracy profiles over a CPA concentration range from 33 to 667 ng/ml for each skin layer. Finally, the method was successfully applied to the determination of CPA to several skin samples after topical application of different gel formulations containing CPA.

Generic systems for the enantioseparation of basic drugs in NACE using single-isomer anionic CDs.

The enantioseparation of 10 basic drugs was evaluated in NACE systems using heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)-ß-CD (HMAS-ß-CD). For this purpose, a D-optimal design with 21 experimental points was applied. Four antifungal agents (econazole, isoconazole, miconazole, sulconazole), three local anesthetics (bupivacaine, mepivacaine and prilocaine), two sympathomimetics (salbutamol and terbutaline) and one ß-blocker (carvedilol) were selected as basic model analytes. The influence on the enantiomeric resolution of anionic CD and BGE anion concentrations as well as the BGE anion nature was investigated. For all studied analytes, the enantiomeric resolution was shown to be significantly influenced by the CD concentration. Based on the observed results, a generic NACE system was recommended, namely 20mM HMAS-ß-CD and 10mM ammonium camphor SO(3)(-) in methanol acidified with 0.75 M formic acid. Moreover, this NACE system was compared to previous conditions with heptakis(2,3-di-O-methyl-6-O-sulfo)-ß-CD (HDMS-ß-CD) or heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD (HDAS-ß-CD). Finally, two generic systems using either HDAS-ß-CD or HMAS-ß-CD were proposed and evaluated for the enantioseparation of ketamine and norketamine after incubation of ketamine in phenobarbital-induced male rat liver microsomes systems.

Pre-study and in-study validation of a SPE-LC-MS-MS method for the determination of 5-S-cysteinyldopa, a melanoma biomarker, in human plasma.

The incidence of malignant melanoma has increased over the past decades, particularly in Caucasian population. This disease presents defavourable prognosis in terms of survey, especially when detection occurs at the metastatic phase. Reliable analytical methods for biomarker determination are thus an interesting tool in pathology detection and follow-up. In this context, a method using SPE-LC-ESI-MS-MS for the determination of 5-S-cysteinyldopa (5-SCD) in human plasma was optimized. The presence of matrix effect was investigated in details while 5-SCD stability was studied according to FDA requirements for the validation of bioanalytical methods. Pre-study and in-study validations of the entire method were then successfully performed by applying the approach based on total measurement error and accuracy profiles over a concentration ranges from 1.6 to 200 ng/ml. Good results with respect to accuracy, trueness and precision were obtained. The maximum risk of observing future measurements falling outside the acceptance limits during routine analysis was also estimated.

Development and validation of a ultra-high-performance liquid chromatography-UV method for the detection and quantification of erectile dysfunction drugs and some of their analogues found in counterfeit medicines.

Pharmaceutical counterfeiting is a permanently growing problem. Control laboratories are constantly analysing counterfeit medicines. In industrialised countries, one of the main counterfeited class of medicines are erectile dysfunction drugs. This paper describes the development and validation of a fast method to detect and quantify the three authorised phosphodiesterase type 5 inhibitors and five analogues. The method is based on the use of a sub-2 microns polar-embedded column with a gradient using acetonitrile as organic modifier and 10mM ammonium formate buffer (pH 3.5) as aqueous component of the mobile phase. The separation was achieved in less than 4.5 min. The method has also been compared to the registered HPLC method for the assay of Viagra(®) which was considered as the reference method. The method is also compatible with on-line coupling mass spectrometry and will significantly reduce analysis times and solvent consumption.

Impurity fingerprints for the identification of counterfeit medicines--a feasibility study.

Most of the counterfeit medicines are manufactured in non good manufacturing practices (GMP) conditions by uncontrolled or street laboratories. Their chemical composition and purity of raw materials may, therefore, change in the course of time. The public health problem of counterfeit drugs is mostly due to this qualitative and quantitative variability in their formulation and impurity profiles. In this study, impurity profiles were treated like fingerprints representing the quality of the samples. A total of 73 samples of counterfeit and imitations of Viagra(®) and 44 samples of counterfeit and imitations of Cialis(®) were analysed on a HPLC-UV system. A clear distinction has been obtained between genuine and illegal tablets by the mean of a discriminant partial least squares analysis of the log transformed chromatograms. Following exploratory analysis of the data, two classification algorithms were applied and compared. In our study, the k-nearest neighbour classifier offered the best performance in terms of correct classification rate obtained with cross-validation and during external validation. For Viagra(®), both cross-validation and external validation sets returned a 100% correct classification rate. For Cialis(®) 92.3% and 100% correct classification rates were obtained from cross-validation and external validation, respectively.