A sensitive, specific, and rapid high‐performance liquid chromatography ( HPLC ) method for the determination of ambrisentan enantiomers has been developed and validated. Six chiral columns were tested in a reversed‐phase system. Excellent enantioseparation with the resolution more than 2.5 was achieved on C hiralcel OZ ‐3 R (cellulose 3‐chloro‐4‐methylphenylcarbamate) using mixture of 20 m M sodium formate (p H 3.0) with acetonitrile (55:45; v/v). Validation of the HPLC method including linearity, limit of detection, limit of quantification, precision, accuracy, and selectivity was performed according to the I nternational C onference on H armonisation ( ICH ) guidelines. The method has an advantage of a very quick chromatographic separation (less than 6 min) and therefore is highly suitable for routine determination of ( R )‐ambrisentan in enantiopure active pharmaceutical ingredient ( S )‐ambrisentan.
A case study on Sitagliptin drug products and Sitagliptin/Metformin drug products concerning contamination with N-nitrosamines was performed using two newly developed analytical methods for determination of N-nitroso-triazolopyrazine (NTTP; 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine) and its precursor triazolopyrazine (3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine) and also several methods published previously. The method for determination of triazolopyrazine uses a liquid chromatograph Acquity H-Class (Waters) coupled with a simple QDa detector (Waters). Separation was achieved on a column Acquity CSH C18 (Waters) using a linear elution gradient with a mobile phase consisting of aqueous ammonium formate buffered at pH 4.2 and acetonitrile. The detection was performed using positive electrospray and selected ion monitoring at m/z 193. The method for ultra-trace determination of NTTP uses a liquid chromatograph Acquity I-Class (Waters) coupled with a tandem mass spectrometer Xevo TQ-XS. Separation of NTTP was achieved on a column Acquity HSS T3 using a linear elution gradient with a mobile phase consisting of 0.1% formic acid in water and methanol. The detection was performed using positive electrospray and multiple reaction monitoring at transitions m/z 222.15→42.05 (collision energy 17 eV) and m/z 222.15→192.15 (collision energy 11 eV). Both methods were used during an optimization study of manufacturing of Sitagliptin and Sitagliptin/Metformin drug products. The goal of the study was to decrease NTTP content in the final drug product under the strict legislative limit set by Federal Drug Agency. The efficacy of several solutions was proven, but could not be fully disclosed due to Intellectual Property Protection policy of Zentiva. Instead, a brief review of recently published strategies to cope with N-nitrosamine contamination is presented.
The retention behavior of several series of free α‐ and ω‐amino acids and positional isomers of amino pentanoic acid in the hydrophilic interaction chromatography mode (HILIC) was studied. The study was carried out on three stationary phases followed by post‐column derivatization with fluorescence detection in order to describe the retention mechanism of the tested amino acids. The effect of chromatographic conditions including acetonitrile content in the mobile phase, mobile phase pH (ranging from 3.5 to 6.5) and concentration of buffer in the mobile phase was investigated. The effect of the number of carbon atoms ( n C ) in aliphatic chains of the individual homologue of α‐ and ω‐amino acids and the logarithm of the partition coefficient (log D ) on retention was also a part of the presented study. A good correlation ( r > 0.98) between the log k and log D values of amino acids or n C , respectively, was observed. The described linear relationships were subsequently applied to predict the retention behavior of individual members of the homologous series of amino acids and to optimize the mobile phase composition in HILIC. The obtained results confirmed that the retention mechanism of α‐amino acids, ω‐amino acids and positional isomers of amino acids was based on the log D values and the number of carbon atoms in the aliphatic chains of amino acids. The elution order of ω‐amino acids and positional isomers of amino pentanoic acid was strongly dependent on the mobile phase pH in the investigated range whereas the retention factors of all α‐amino acids remained essentially unchanged on all tested stationary phases.
An improved and scalable process for the production of montelukast (Singulair, drug for asthma) based on a new and advantageous method of carrying out the key substitution reaction has been developed. The present procedure is distinguished from the previous solutions in the use of linear or cyclic polyethers, which ensures higher selectivity of the key step. The improved process for the preparation of montelukast is able to minimize a content of impurities and allows the effective production of montelukast and its scale-up.
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