The effect of the composition of a fixed dose combination on bioequivalence results.

The purpose of this work was to develop a new supergeneric product Meloxicam/Omeprazole. Such a combination brings a benefit in terms of decreasing side effects for the patients using meloxicam. The new combination is composed of a meloxicam powder blend (MPB) and omeprazole gastro-resistant pellets (OAP) in hard gelatin capsules. The main tasks were to select the excipients to keep the functional layer of OAP active and to prove the bioequivalence to the original products of meloxicam tablets together with omeprazole capsules. Although dissolution profiles similar to the original product were obtained, the unexpected results of omeprazole low bioavailability in the fed bioequivalence study (BES I) showed the necessity to investigate the formulation in greater depth. A modified more complex dissolution method was developed in order to understand the release of omeprazole under gastric conditions. This method revealed the degradation of omeprazole in the formulation when exposed to the fed conditions because of the increase in microenvironmental pH in the capsule caused by trisodium citrate, commonly used for improving solubility of meloxicam. This pH increase dissolved the gastro-resistant layer of OAP and caused the chemical degradation. To prevent this effect, a trisodium citrate-free formulation was developed. Reformulated capsules passed the repeated fed bioequivalence study (BES II).

Esterification of Ibuprofen in Soft Gelatin Capsules Formulations-Identification, Synthesis and Liquid Chromatography Separation of the Degradation Products.

Unknown impurities were identified in ibuprofen (IBU) soft gelatin capsules (SGCs) during long-term stability testing by a UHPLC method with UV detection and its chemical formula was determined using high resolution/accurate mass (HRAM) LC-MS. Reference standards of the impurities were subsequently synthesized, isolated by semi-preparative HPLC and characterized using HRAM LC-MS, NMR and IR. Two impurities were formed by esterification of IBU with polyethylene glycol (PEG), which is used as a fill of the SGCs, and were identified as IBU-PEG monoester and IBU-PEG diester. Two other degradants arised from reaction of IBU with sorbitol and sorbitan, which are components of the shell and serves as plasticizers. Thus, IBU sorbitol monoester (IBU-sorbitol) and IBU sorbitan monoester (IBU-sorbitan ester) were identified. An UHPLC method was further optimized in order to separate, selectively detect and quantify the degradation products in IBU SGCs.

Utilization of Photochemically Induced Fluorescence Detection for HPLC Determination of Genotoxic Impurities in the Vortioxetine Manufacturing Process.

An analytical reversed-phase high-performance liquid chromatography (HPLC) method for the detection and quantitative determination of two genotoxic impurities at ppm level present in the vortioxetine manufacturing process is described. Applying the concept of threshold of toxicological concern, a limit of 75 ppm each for both genotoxic impurities was calculated based on the maximum daily dose of active pharmaceutical ingredients. The novel reversed-phase HPLC method with photochemically induced fluorescence detection was developed on XSELECT Charged Surface Hybrid Phenyl-Hexyl column using the mobile phase consisted a mixture of 10 mM ammonium formate pH 3.0 and acetonitrile. The elution was performed using an isocratic composition of 48:52 (v/v) at a flow rate of 1.0 mL/min. The photochemically induced fluorescence detection is based on the use of UV irradiation at 254 nm through measuring the fluorescence intensity at 300 nm and an excitation wavelength of 272 nm to produce fluorescent derivatives of both genotoxic impurities. The online photochemical conversion and detection is easily accomplished for two expected genotoxic impurities and provides a sufficiently low limit detection and quantification for the target analysis.

HILIC-MS Determination of Genotoxic Impurity of 2-Chloro-N-(2-Chloroethyl)Ethanamine in the Vortioxetine Manufacturing Process.

In the last decade, pharmaceutical regulatory agencies are focused on monitoring and evaluation of trace-level genotoxic impurities (GTIs) in drug substances, which requires manufacturers to deliver innovative approaches for their analysis and control. GTIs in the low p.p.m. level rising from the process of drug production have to be positively identified and quantified. Therefore, sensitive and selective analytical methods are necessary for required quantification level of these GTIs. Unfortunately, general guidance on how to develop strategy of the analysis and control of GTIs is currently missing in the pharmaceutical industry. Therefore, practical example of the analytical control of 2-chloro-N-(2-chloroethyl)ethanamine GTI in the vortioxetine (VOR) manufacturing process was demonstrated in this work. QDa mass detection with electrospray ionization in selected-ion recording mode was utilized for quantitation of GTIs. The method of hydrophilic interaction liquid chromatography coupled with mass spectrometry detection (HILIC-MS) was validated as per International Conference on Harmonization guidelines and was able to quantitate GTIs at 75 p.p.m. with respect to VOR. The HILIC-MS method was achieved using a Primesep B column (150 × 4.6 mm, 5.0 µm; Sielc, USA) using mobile phase consisting of 10 mM ammonium formate buffer pH 3.0 and acetonitrile (5 : 95, v/v) at 0.8 mL/min flow rate. The QDa mass detector was operated in the positive ion mode. Quadrupole mass analyzer was employed in selected-ion monitoring mode using target ion at m/z 142 as [M+H](+).

Direct analysis in real time--high resolution mass spectrometry as a valuable tool for the pharmaceutical drug development.

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.

Retention behavior of a homologous series and positional isomers of aliphatic amino acids in hydrophilic interaction chromatography.

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 (nC) in aliphatic chains of the individual homologue of α- and ω-amino acids and the logarithm of the partition coefficient (logD) on retention was also a part of the presented study. A good correlation (r > 0.98) between the logk and logD values of amino acids or nC, 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 logD 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.

Identification, characterization, synthesis and HPLC quantification of new process-related impurities and degradation products in retigabine.

Two new impurities were described and determined using gradient HPLC method with UV detection in retigabine (RET). Using LC-HRMS, NMR and IR analysis the impurities were identified as RET-dimer I: diethyl {4,4'-diamino-6,6'-bis[(4-fluorobenzyl)amino]biphenyl-3,3'-diyl}biscarbamate and RET-dimer II: ethyl {2-amino-5-[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino] phenyl}carbamate. Reference standards of these impurities were synthesized followed by semipreparative HPLC purification. The mechanism of the formation of these impurities is also discussed. An HPLC method was optimized in order to separate, selectively detect and quantify all process-related impurities and degradation products of RET. The presented method, which was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and selectivity is very quick (less than 11min including re-equilibration time) and therefore highly suitable for routine analysis of RET related substances as well as stability studies.

Chip-based nano-LC-MS/MS identification of proteins in complex biological samples using a novel polymer microfluidic device.

Although the proteome of each organism is unambiguously coded in its genome, the proteome shows the real biology in action in each particular organism. New powerful tools are being developed for biochemists and biologists to analyze complex biological samples for studying the complete protein supplement of the genome, i. e., the proteome. There are several methods available for proteome analysis including 2-DE and several forms of MS. In recent years, technologies such as microfluidics and array-based systems have appeared in the field of analysis, identification, and quantification of proteins. These novel approaches might help in solving current technical challenges in proteomics. This paper presents a practical application of the first commercially available microfluidic nano-ESI device coupled with nano-LC (i. e., HPLC-chip) for the analysis of samples of some biological protein mixtures.

System peaks and their positive and negative aspects in chromatographic techniques.

Whenever a mobile phase contains more than one component, additional signals commonly called system peaks can appear. The origin of these signals is explained through loss of equilibrium in the separation column caused by injection of analyte dissolved in a different solvent than the mobile phase. The system peaks are then generated by a relaxation process started by the non-equilibrium state. An overview of the theory and applications of the system peaks in separation methods, mainly in liquid chromatography, is presented in this paper. Only a brief theoretical discussion of the system peak origin is given as the theoretical aspects of system peak formation have already been published in many papers. The main focus of this review is to summarize applications, in which system peaks were used to measure physical or physicochemical data. Signals of system peaks are often misinterpreted but they offer valuable information about thermodynamics and kinetics of the separation process that takes place in chromatographic column.