Synthesis and Physicochemical Characterization of the Process-Related Impurities of Olmesartan Medoxomil. Do 5-(Biphenyl-2-yl)-1-triphenylmethyltetrazole Intermediates in Sartan Syntheses Exist?

During the process development for multigram-scale synthesis of olmesartan medoxomil (OM), two principal regioisomeric process-related impurities were observed along with the final active pharmaceutical ingredient (API). The impurities were identified as N-1- and N-2-(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl derivatives of OM. Both compounds, of which N-2 isomer of olmesartan dimedoxomil is a novel impurity of OM, were synthesized and fully characterized by differential scanning calorimetry (DSC), infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry/electrospray ionization (HRMS/ESI). Their ¹H, (13)C and (15)N nuclear magnetic resonance signals were fully assigned. The molecular structures of N-triphenylmethylolmesartan ethyl (N-tritylolmesartan ethyl) and N-tritylolmesartan medoxomil, the key intermediates in OM synthesis, were solved and refined using single-crystal X-ray diffraction (SCXRD). The SCXRD study revealed that N-tritylated intermediates of OM exist exclusively as one of the two possible regioisomers. In molecular structures of these regioisomers, the trityl substituent is attached to the N-2 nitrogen atom of the tetrazole ring, and not to the N-1 nitrogen, as has been widely reported up to the present. This finding indicates that the reported structural formula of N-tritylolmesartan ethyl and N-tritylolmesartan medoxomil, as well as their systematic chemical names, must be revised. The careful analysis of literature spectroscopic data for other sartan intermediates and their analogs with 5-(biphenyl-2-yl)tetrazole moiety showed that they also exist exclusively as N-2-trityl regioisomers.

Development and validation of high performance liquid chromatography with a spectrophotometric detection method for the chemical purity and assay of nepafenac.

The study is a proposition of the application of high performance liquid chromatography (HPLC) with a spectrophotometric UV range detector to analyze the chemical purity and assay of nepafenac, an active pharmaceutical ingredient (API). During literature search only a few publications were found about nepafenac. HPLC UV methods were mainly presented in patent documents about nepafenac synthesis and chemical purity. The presented method allows to separate all potential related compounds from nepafenac and to quantitate the nepafenac amount. As there is no official monograph in the pharmacopeias about nepafenac, the performed full validation procedure makes the method ready to use in routine analysis. The composition of the mobile phase (10mM ammonium formate, pH 4.1) and the HPLC column (Phenomenex Gemini-NX C18) were selected during the development step. Presented data confirm the benefits of the developed method. Four of the most potential impurities were validated as for the quantitative test and the rest of impurities were validated as for the limit test - according to ICH Q2(R1). The accuracy/recovery results for the chemical purity method are within 90-108%, in the case of assay studies from 99% to 101%; the limit of detection is as low as 15-30ng/mL. The linearity passes all statistical tests.

Stability of sample solution as a crucial point during HPLC determination of chemical purity of temozolomide drug substance.

An HPLC method for determination of related substances in temozolomide drug substance was developed. Particular attention was paid to the stability studies due to the fact that temozolomide is unstable in a solution and quickly decomposes to its main degradation product 5-amino-4-imidazolecarboxamide (AIC). A mixture of diluted acetic acid and acetonitrile (4:1, v/v) as a diluent guaranteed lowering the decomposition of temozolomide in the solution. As it is not practically possible to fully eliminate the decomposition of temozolomide during an analysis, the mathematical correction of the results was proposed which allows to analyse almost five times more samples per week, comparing to the procedure without the application of the correction. The accuracy of the correction procedure was proved by investigating the recovery of AIC spiked to temozolomide solutions at different levels. Recoveries equalled 90-108% for AIC concentrations contained in the range of 0.30-1.80 µg ml(-1). The developed method was validated according to the current guidelines, proving the suitability of the method for its intended purpose.

Blue natural organic dyestuffs--from textile dyeing to mural painting. Separation and characterization of coloring matters present in elderberry, logwood and indigo.

Natural dyestuffs used for painting or dyeing of textiles are complex mixtures of compounds of various chemical properties. Proper identification of the dye used by a painter and, even better, its origin is possible only when its compositional 'fingerprint' can be evaluated. For this reason gradient program for liquid chromatographic separation of 16 color compounds--components of natural blue dyes: elderberry, logwood and indigo--has been developed. Two detector systems were used simultaneously: UV-Vis spectrophotometry (at 280, 445, 520 and 600 nm) and ESI mass spectrometry (positive and negative SIM mode). It was found that fragmentation observed in ESI-MS is affected not only by ion source parameters, but also by chromatographic conditions, especially in case of the less stable substances: cyanidin glucosides, tannic acid, rutin and hematoxylin. Examination of characteristic dissociation pathways of the compounds under investigation after direct admission into ion source or after chromatographic separation allowed to select proper ions for SIM detection and to develop novel and efficient reversed phase high performance liquid chromatographic (RP-HPLC)-UV-Vis/ESI-MS method for the analysis of natural blue dyes. The procedure was successfully applied for identification of indigotin and carminic acid-main colorants extracted from a fiber taken from the blue-red 'Italian' tapestry (the collection of the National Museum in Warsaw, Poland).

Identification of indigoid dyes in natural organic pigments used in historical art objects by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry.

Pigments are among the most important components of historical paintings and textiles and their nature provides the unique character of color. They can be divided into two main groups: inorganic and organic, extracted from plants or animals. Their identification is a necessary stage in the conservation of art objects. Reversed-phase liquid chromatography with electrospray ionization mass spectrometry (ESI-MS) and UV/visible spectrophotometric methods were elaborated for the identification of indigoid (indigo, indirubin, isoindigo, isoindirubin) color components of natural dyestuffs and their natural or synthetic precursors (indican, isatin, indoxyl, 2-indolinone). ES-MS offers detection limits in the range 0.03-5.00 microg ml(-1) for the color compounds examined. The method developed made it possible to identify indigo and its isomers in genuine Indian indigo, indigo from woad and Tyrian Purple. It was applied to the identification of natural dyes on fiber from a 19th century Japanese tapestry, 'Cranes in the landscape'. A procedure based on freezing and grinding of a sample before the extraction of dyes from the textile was developed. The components of the extract obtained were identified after acidic hydrolysis as indigotin and methylene blue.

Identification of anthraquinone coloring matters in natural red dyes by electrospray mass spectrometry coupled to capillary electrophoresis.

Capillary electrophoresis with UV/visible diode-array detection (DAD) and electrospray mass spectrometric (ESI-MS) detection were used for the identification of anthraquinone color components of cochineal, lac-dye and madder, natural red dyestuffs often used by ancient painters. For the purpose of such analysis, ESI-MS was found to be a much more appropriate detection technique than DAD one owing to its higher sensitivity (detection limits in the range 0.1-0.5 micro g ml(-1)) and selectivity. The method developed made it possible to identify unequivocally carminic acid and laccaic acids A, B and E as coloring matters in the examined preparations of cochineal and lac-dye, respectively. In madder, European Rubia tinctorum, alizarin and purpurin were found. The method allows the rapid, direct and straightforward identification and quantification of components of natural products used in art and could be very helpful in restoration and conservation procedures.