A solvent-modified micellar electrokinetic chromatography method was developed following the Quality by Design approach for the simultaneous determination of sitagliptin (SIT), an oral antihyperglycemic drug, and its main impurities derived from the synthesis process. The separation system was identified in the scouting phase and was made by sodium dodecyl sulphate (SDS) micelles with the addition of n-butanol and methanol. The knowledge space was investigated through an asymmetric screening matrix, taking into consideration eight critical method parameters (CMPs) involving the composition of the background electrolyte in terms of buffer concentration and pH, the concentration of surfactants and organic modifiers, and voltage. The critical method attributes (CMAs) were identified as analysis time and the distance between the tail of the electroosmotic flow system peak and the front edge of impurity I1 (sitagliptin triazole hydrochloride). A Box-Behnken Design was used in response surface methodology for calculating the quadratic models relating the CMPs to the CMAs. From the models it was possible to compute the method operable design region (MODR) through Monte-Carlo simulations. The MODR was identified in the probability maps as the multidimensional zone where the risk of failure to achieve the desired values for the CMAs was lower than 10 %. The experimental conditions corresponding to the working point, with the MODR interval, were the following: background electrolyte, 14 (10-18) mM borate buffer pH 9.20, 100 mM SDS, 13.6 (11.1-16.0) %v/v n-butanol, 6.7 (4.5-8.8) %v/v methanol; voltage and temperature were set to 28 kV and 22 °C, respectively. The developed CE method was validated in accordance with International Council for Harmonisation guidelines and was applied to the analysis of SIT tablets. The routine analysis for the quality control of the pharmaceutical product could be conducted in about 11 min.

• Klíčová slova
• Capillary electrophoresis, Experimental design, Impurities, Method operable design region, Quality by design, Sitagliptin,
• MeSH
• chromatografie micelární elektrokinetická kapilární * MeSH
• dodecylsíran sodný MeSH
• micely * MeSH
• reprodukovatelnost výsledků MeSH
• rozpouštědla MeSH
• sitagliptin fosfát MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dodecylsíran sodný MeSH
• micely * MeSH
• rozpouštědla MeSH
• sitagliptin fosfát MeSH

A capillary electrophoresis method for the simultaneous determination of the enantiomeric purity and of impurities of the chiral calcimimetic drug cinacalcet hydrochloride has been developed following Quality by Design principles. The scouting phase was aimed to select the separation operative mode and to identify a suitable chiral selector. Among the tested cyclodextrins, (2-carboxyethyl)-β-cyclodextrin and (2-hydroxypropyl)-γ-cyclodextrin (HPγCyD) showed good chiral resolving capabilities. The selected separation system was solvent-modified capillary zone electrophoresis with the addition of HPγCyD and methanol. Voltage, buffer pH, methanol concentration and HPγCyD concentration were investigated as critical method parameters by a multivariate strategy. Critical method attributes were represented by enantioresolution and analysis time. A Box-Behnken Design allowed the contour plots to be drawn and quadratic and interaction effects to be highlighted. The Method Operable Design Region (MODR) was identified by applying Monte-Carlo simulations and corresponded to the multidimensional zone where both the critical method attributes fulfilled the requirements with a desired probability π≥90%. The working conditions, with the MODR limits, corresponded to the following: capillary length, 48.5cm; temperature, 18°C; voltage, 26kV (26-27kV); background electrolyte, 150mM phosphate buffer pH 2.70 (2.60-2.80), 3.1mM (3.0-3.5mM) HPγCyD; 2.00% (0.00-8.40%) v/v methanol. Robustness testing was carried out by a Plackett-Burman matrix and finally a method control strategy was defined. The complete separation of the analytes was obtained in about 10min. The method was validated following the International Council for Harmonisation guidelines and was applied for the analysis of a real sample of cinacalcet hydrochloride tablets.

• Klíčová slova
• Capillary electrophoresis, Chiral separation, Cinacalcet, Impurities, Method operable design region, Quality by Design,
• MeSH
• beta-cyklodextriny chemie   MeSH
• cinakalcet chemie   izolace a purifikace   MeSH
• elektroforéza kapilární metody   MeSH
• gama-cyklodextriny chemie   MeSH
• hodnocení rizik MeSH
• koncentrace vodíkových iontů MeSH
• kontaminace léku MeSH
• metoda Monte Carlo MeSH
• pravděpodobnost MeSH
• rozpouštědla MeSH
• stereoizomerie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-cyklodextriny MeSH
• betadex MeSH Prohlížeč
• cinakalcet MeSH
• gama-cyklodextriny MeSH
• gamma-cyclodextrin MeSH Prohlížeč
• rozpouštědla MeSH

An HPLC method with UV and electrospray ionization - mass spectrometry (ESI-MS) detection was developed for the separation and determination of obeticholic acid (OBE) and its related compounds in development process and quality control. OBE and its related compounds were classified into three major group based on the mass spectra profiles: (A) those containing a hydroxyl group at position 3 and 7, (B) those containing a hydroxyl group and/or carbonyl group at position 3, hydrogen, ethyl or ethylidene group at position 6 and a hydroxyl group and/or carbonyl group at position 7, and (C) those containing carbonyl groups at position 3 and 7. ESI-MS ionization of OBE and its related compounds often produced intense adduct ions [M+H+98]+ and/or [M+H+196]+ that were identified as the adduct ions of phosphoric acid ([M+H+H3PO4]+ and [M+H+2H3PO4]+) originating from the mobile phase. The separation on HPLC system was accomplished using stationary phase based on XSelect CSH C18 (3.0×150mm×2.5μm) and a linear gradient elution using acetonitrile and 0.05% of o-phosphoric acid. The condition of chromatographic system was set as follows: flow rate 0.7mL/min, temperature 45°C and UV detection at 192nm. The separation of the 19 compounds was finished in less than 18min (including equilibration time). The HPLC/UV method was partially validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines in terms of repeatability, selectivity, linearity and limit of quantification and detection.

• Klíčová slova
• LC separation, Mass adducts with phosphoric acid, Mass detection, Obeticholic acid,
• MeSH
• chemická frakcionace přístrojové vybavení   metody   MeSH
• chemie farmaceutická metody   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací přístrojové vybavení   metody   MeSH
• kontaminace léku prevence a kontrola   MeSH
• kyselina chenodeoxycholová analogy a deriváty   analýza   chemie   MeSH
• limita detekce MeSH
• příprava léků přístrojové vybavení   metody   MeSH
• reprodukovatelnost výsledků MeSH
• řízení kvality * MeSH
• senzitivita a specificita MeSH
• spektrofotometrie ultrafialová přístrojové vybavení   metody   MeSH
• vysokoúčinná kapalinová chromatografie přístrojové vybavení   metody   MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH
• Názvy látek
• kyselina chenodeoxycholová MeSH
• obeticholic acid MeSH Prohlížeč

A capillary electrophoresis method for the simultaneous determination of the enantiomeric purity and of impurities of the chiral drug ambrisentan has been developed following the Quality by Design principles. The selected separation system consisted of a micellar pseudostationary phase made by sodium dodecyl sulphate with the addition of γ-cyclodextrin. The effects of critical process parameters (capillary length, temperature, voltage, borate concentration, pH, sodium dodecyl sulphate concentration, γ-cyclodextrin concentration) on enantioresolution of ambrisentan and analysis time were extensively investigated by multivariate strategies involving a screening phase and Response Surface Methodology. The Design Space was defined with a desired probability level π≥90%, and the working conditions, with the limits of the Design Space, corresponded to the following: capillary length, 64.5cm; temperature, 22°C; voltage, 30kV (26-30kV); background electrolyte, 100mM borate buffer pH 9.20 (8.80-9.60), 100mM sodium dodecyl sulphate, 50mM (43-50mM) γ-cyclodextrin. A Plackett-Burman design was applied for robustness testing, and a method control strategy was established. The method was fully validated according to the International Conference on Harmonisation guidelines and was applied to ambrisentan coated tablets.

• Klíčová slova
• Ambrisentan, Capillary electrophoresis, Chiral separation, Experimental design, Impurities, Quality by design,
• MeSH
• boritany chemie   MeSH
• chromatografie micelární elektrokinetická kapilární * metody   MeSH
• cyklodextriny MeSH
• dodecylsíran sodný MeSH
• fenylpropionáty analýza   chemie   MeSH
• kalibrace MeSH
• koncentrace vodíkových iontů MeSH
• kontaminace léku MeSH
• pufry MeSH
• pyridaziny analýza   chemie   MeSH
• reprodukovatelnost výsledků MeSH
• řízení kvality MeSH
• stereoizomerie MeSH
• tablety analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ambrisentan MeSH Prohlížeč
• boritany MeSH
• cyklodextriny MeSH
• dodecylsíran sodný MeSH
• fenylpropionáty MeSH
• pufry MeSH
• pyridaziny MeSH
• tablety MeSH

A novel and sensitive derivatization procedure for the determination of 2-cynaoacetamide in pharmaceutical samples using liquid chromatography with the fluorescence detection was discovered. The method is based on derivatization of 2-cynaoacetamide using 2-hydroxyacetophenone as a new derivatization reagent. The product of derivatization reaction was isolated and characterized using spectroscopic techniques namely LC-MS, NMR and IR. The structure of 2-cyanoacetamide derivative was unambiguously assigned as a 2-amino-4-phenylfuran-3-carboxamide. Two derivatization systems were optimized in terms of reaction temperature, reaction time, pH and concentration of 2-hydroxyacetophenone, and a new pre- and post-derivatization HPLC methods were developed. The separations on HPLC with pre-column derivatization were accomplished using stationary phase based on a XBridge C18 column (100×4.6, 3.5μm) and isocratic elution using the mobile phase acetonitrile - 0.1% formic acid (30:70, v/v). The separations on the HPLC with post-column derivatization were performed on stationary phase on a TSKgel Amide-80 column (150×4.6mm, 3μm). The mobile phase was a mixture of acetonitrile, methanol and 10mM sodium formate buffer at pH=4.5 in ratio 3:2:95 (v/v). Both HPLC methods were fully validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy and precision according to ICH guidelines. The pre-column derivatization method was linear in the range 1.1-2000μg/l with method accuracy≥98.2% and method precision RSD≤4.8%. The post-column derivatization method was linear in the range 12-2000μg/l. Method accuracy≥96.3% and method precision RSD≤3.5%. Proposed new methods were proved to be highly sensitive, simple and rapid, and were successfully applied to the determinations of 2-cynaoacetamide in pregabalin.

• Klíčová slova
• 2-cyanoacetamide, 2-hydroxyacetophenone, Derivatization, Fluorescence detection, Pregabalin,
• MeSH
• acetofenony chemie   MeSH
• hmotnostní spektrometrie MeSH
• indikátory a reagencie * MeSH
• magnetická rezonanční spektroskopie MeSH
• nitrily analýza   MeSH
• pregabalin chemie   MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH
• Názvy látek
• 2-cyanoacetamide MeSH Prohlížeč
• 2'-hydroxyacetophenone MeSH Prohlížeč
• acetofenony MeSH
• indikátory a reagencie * MeSH
• nitrily MeSH
• pregabalin MeSH

Agomelatine is one of the newest antidepressants. Due to a different mechanism of action it offers a completely new approach in the treatment of depressive disorders. Two chromatographic methods for determination of agomelatine and its impurities were developed. The separations on UHPSFC system were accomplished using stationary phase based on BEH 2-EP and gradient elution with CO2 and methanol containing 20mM ammonium formate and 5% of water. The UHPLC separations were performed on stationary phase BEH Shield RP18. The mixture of acetonitrile and methanol in ratio 1:1 and ammonium acetate buffer pH 9.5 were used as mobile phase. Both developed methods were properly validated in terms of linearity, sensitivity (LOD, LOQ), accuracy and precision according to ICH guidelines. The UHPSFC method was linear in the range 0.25-70μg/ml for all analytes with method accuracy ≥97.4% and ≥100.2% and method intra-day precision RSD ≤2.4 and ≤0.8 for impurities and API (active pharmaceutical ingredient), respectively. The UHPLC method was linear in the range 0.1-10μg/ml for all analytes except three impurities for which the linear range was larger 0.1-25μg/ml. Method accuracy ≥95.7% and ≥95.2% and method intra-day precision RSD ≤2.6 and ≤1.5 were found for impurities and API, respectively. The measurement of tablet samples was performed and the selected parameters of the methods were compared. In conclusion, both methods were appropriate for the determination of agomelatine and its impurities in pharmaceutical quality control (QC), although the UHPSFC method was found as more convenient especially in the method development phase. The advantages of newly developed UHPSFC-PDA (photo diode array detector) method were its environmental friendliness due to the mobile phase used, a very good resolution, selectivity and high speed of analysis (total time of separation was 4.1min).

• Klíčová slova
• Agomelatine, Method development, UHPLC, UHPSFC, Ultra-high performance liquid chromatography, Ultra-high performance supercritical fluid chromatography,
• MeSH
• acetamidy analýza   MeSH
• koncentrace vodíkových iontů MeSH
• referenční standardy MeSH
• tablety MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• validační studie MeSH
• Názvy látek
• acetamidy MeSH
• agomelatine MeSH Prohlížeč
• tablety MeSH

Internal normalization (IN) serves as a quantitative tool in gas chromatography. Nevertheless, its utilization in liquid chromatography is not widely employed, as several requirements need to be taken into account. However, IN can be used in case of relative amounts estimation when the absolute concentration is not the crucial factor. This suits very well in pharmaceutical analysis when the relative amount of active pharmaceutical ingredient (API) impurities is to be estimated with a limited knowledge of statistics, such as t-test and linear regression. The determination of three prasugrel impurities in the real sample by means of IN and the comparison of these results with external standard calibration was presented. The IN method was validated by test of population means and variances agreement and the agreement of external calibration and IN was performed by Student t-test. The influence of impurities concentration above and below is also discussed as well as the validation parameters, LOD and LOQ. It was found that the results achieved by external calibration and IN are statistically the same and, therefore, IN is a proper method for relative amount estimation of API impurities.

• Klíčová slova
• Internal normalization, Pharmaceutical substances, Quantification, Validation,
• MeSH
• chemie farmaceutická normy   MeSH
• kalibrace MeSH
• koncentrace vodíkových iontů MeSH
• léčivé přípravky chemie   MeSH
• limita detekce MeSH
• prasugrel hydrochlorid analýza   MeSH
• reprodukovatelnost výsledků MeSH
• vysokoúčinná kapalinová chromatografie metody   normy   MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH
• Názvy látek
• léčivé přípravky MeSH
• prasugrel hydrochlorid MeSH

In this study, direct analysis in real time-mass spectrometry (DART-MS) was assessed for the analysis of various pharmaceutical formulations with intention to summarize possible applications for the routine pharmaceutical development. As DART is an ambient ionization technique, it allows direct analysis of pharmaceutical samples in solid or liquid form without complex sample preparation, which is often the most time-consuming part of the analytical method. This makes the technique suitable for many application fields, including pharmaceutical drug development. DART mass spectra of more than twenty selected tablets and other common pharmaceutical formulations, i.e. injection solutions, ointments and suppositories developed in the pharmaceutical industry during several recent years are presented. Moreover, as thin-layer chromatography (TLC) is still very popular for the monitoring of the reactions in the synthetic chemistry, several substances were analyzed directly from the TLC plates to demonstrate the simplicity of the technique. Pure substance solutions were spotted onto a TLC plate and then analyzed with DART without separation. This was the first DART-MS study of pharmaceutical dosage forms using DART-Orbitrap combination. The duration of sample analysis by the DART-MS technique lasted several seconds, allowing enough time to collect sufficient number of data points for compound identification. The experimental setup provided excellent mass accuracy and high resolution of the mass spectra which allowed unambiguous identification of the compounds of interest. Finally, DART mass spectrometry was also used for the monitoring of the selected impurity distribution in the atorvastatin tablets. These measurements demonstrated DART to be robust ionization technique, which provided easy-to-interpret mass spectra for the broad range of compounds. DART has high-throughput potential for various types of pharmaceutical analyses and therefore eliminates the time for sample cleanup and chromatographic separation.

• Klíčová slova
• DART mass spectrometry, Impurities, Pharmaceutical formulation,
• MeSH
• chromatografie na tenké vrstvě metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• léčivé přípravky analýza   chemie   MeSH
• objevování léků * MeSH
• pomocné látky chemie   MeSH
• tablety chemie   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• léčivé přípravky MeSH
• pomocné látky MeSH
• tablety MeSH