Identification, synthesis and structural characterization of process related and degradation impurities of acrivastine and validation of HPLC method.

Four impurities (Imp-I-IV) were detected using gradient HPLC method in few laboratory batches of acrivastine in the level of 0.03-0.12% and three impurities (Imp-I-III) were found to be known and one (Imp-IV) was unknown. In forced degradation study, the drug is degraded into four degradation products under oxidation and photolytic conditions. Two impurities (Imp-III and -IV) were concurred with process related impurities whereas Imp-V and -VI were identified as new degradation impurities. Based on LC-ESI/MSn study, the chemical structures of new impurities were presumed as 1-[(2E)-3-(4-methylphenyl)-3-{6-[(1E)-3-oxobut-1-en-1-yl]pyridin-2-yl}prop-2-en-1-yl]pyrrolidin-1-ium-1-olate (Imp-IV), 1-{[3-(4-methylphenyl)-3-{6-[(1E)-3-oxobut-1-en-1-yl]pyridin-2-yl}oxiran-2-yl]methyl}pyrrolidin-1-ium-1-olate (Imp-V) and 2-[2-(4-methylphenyl)-3-[(1-oxidopyrrolidin-1-ium-1-yl)methyl]oxiran-2-yl]-6-[(1E)-3-oxobut-1-en-1-yl]pyridin-1-ium-1-olate (Imp-VI), and confirmed by their synthesis followed by spectroscopic analysis, IR, NMR (1H, 13C) and mass. An efficient and selective high-performance liquid chromatography method has been developed and resolved well the drug related substances on a Phenomenex Gemini C-18 (250×4.6mm, particle size 5µm) column. The mobile phase was composed of sodium dihydrogen phosphate (10mM) and methanol, temperature at 25°C, and a PDA detector set at 254nm used for detection. The method was validated with respect to specificity, linearity, precision, accuracy, and sensitivity and satisfactory results were achieved. Identification, synthesis, characterization of impurities and method validation were first reported in this paper.

Determination of five potential genotoxic impurities in dalfampridine using liquid chromatography.

A sensitive and selective HPLC method was developed for identification and quantification of five Potential genotoxic impurities (PGIs) viz. Impurity-I, Impurity-II, Impurity-III, Impurity-IV and Impurity-V in Dalfampridine (Drug substance). The method utilizes Zorbax silica column (250mm×4.6mm, 5.0µm) with UV detector in HILIC (Hydrophilic Interaction Liquid Chromatography) mode for quantitation of five PGIs. It has been validated as per International Council for Harmonisation (ICH) guidelines and is able to quantitate all PGIs at 75ppm with respect to 20mg/mL of sample concentration. It is linear in the range of 22.5-112.5ppm for all PGIs, which matches the range of LOQ-150% of estimated permitted level (75ppm). Its accuracy was established in the range from 88.14 to 107.65% for these PGIs. The correlation coefficient of each impurity was >0.999. It is a good quality control tool for quantitation of PGIs in Dalfampridine at low level.

Simple Isocratic HPLC Method for Determination of Enantiomeric Impurity in Besifloxacin Hydrochloride.

Besifloxacin is a unique chiral broad-spectrum flouroquinolone used in the treatment of bacterial conjunctivitis. R-form of besifloxacin hydrochloride shows higher antibacterial activity as compared to the S-isomer. Therefore, it is necessary to establish chiral purity. To establish chiral purity a high-performance liquid chromatography (HPLC) method for determination of R-besifloxacin and S-besifloxacin (BES impurity A) was developed and validated for in-process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), and lower limit of quantification (LOQ) were determined according to International Council for Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on Chiralpak AD-H (250 x 4.6 mm, 5 µm) column using n-heptane: ethanol: ethylenediamine: acetic acid (800:200:0.5:0.5) (v/v/v/v) as the mobile phase in an isocratic elution. The eluents were monitored by UV/Visible detector at 290 nm. The resolution between S-isomer and besifloxacin hydrochloride was more than 2.0. Based on a signal-to-noise ratio of 3 and 10 the LOD of besifloxacin was 0.30 µg/mL, while the LOQ was 0.90 µg/mL. The calibration curves were linear in the range of 0.9-7.5 µg/mL. Precision of the method was established within the acceptable range. The method was suitable for the quality control enantiomeric impurity in besifloxacin hydrochloride. Chirality 28:628-632, 2016. © 2016 Wiley Periodicals, Inc.

Identification of oxidative degradation impurities of olanzapine drug substance as well as drug product.

Impurities found in stressed and stability studies of olanzapine (polymorphic form-I) [1-7] in both drug substance and drug product are described. These impurities are identified as 4-(4-methyl-1-piperazinyl)-3-hydroxymethylidene-1H-benzo[b][1,4]diazepine-2(3H)-thione (hydroxymethylidene thione) and (Z)-4-(4-methyl-1-piperazinyl)-3-acetoxymethylidene-1H-benzo[b][1,4]diazapine-2(3H)-thione (acetoxymethylidene thione). An oxidative degradation pathway of olanzapine, for the formation of these impurities, has been proposed.

Retinal histopathology of an XLRP carrier with a mutation in the RPGR exon ORF15.

X-linked retinitis pigmentosa comprises the severe forms of RP, with early onset of night blindness, rapid constriction of visual fields and eventual loss of central acuity. Of the five distinct XLRP loci identified on the X chromosome, mutations have been found only in the RP2 and RPGR genes. Of these, mutations in RPGR are more common, particularly in a mutational hot spot that was identified in the newly discovered exon ORF15. We report on an extended family with a microdeletion in RPGR exon ORF15 and the retinal histopathology of a female carrier of this mutation. We found a 1bp deletion at position 632 in exon ORF15 in affected members of family XLRP-319. This mutation alters the reading frame of the predicted RPGR protein, resulting in a premature stop codon. The mutation segregated with disease in three generations of the family and was associated with severe early onset retinal disease in affected men. The retina from a 75 year old carrier female donor had slight photoreceptor loss in the less diseased areas. More severe atrophy with retinal pigment epithelium (RPE) migration was present in areas of the mid- and far periphery. By immunocytochemistry, loss of rhodopsin labelling in rods was found in the areas of focal atrophy and loss of uniform cone spacing was apparent even in well preserved regions. Small multifocal areas of outer retinal degeneration were present in the better preserved regions of the eye. In these foci, rod and cone loss did not coincide. The dissociation of rod and cone degeneration in areas of focal disease is consistent with random X-inactivation early in embryonic development and the occurrence of distinct patterns of radial (rod) and tangential (cone) dispersion during clonal expansion early in photoreceptor differentiation.

Remapping of the RP15 locus for X-linked cone-rod degeneration to Xp11.4-p21.1, and identification of a de novo insertion in the RPGR exon ORF15.

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe, because of their early onset, often leading to significant vision loss before the 4th decade. Previously, the RP15 locus was assigned to Xp22, by linkage analysis of a single pedigree with "X-linked dominant cone-rod degeneration." After clinical reevaluation of a female in this pedigree identified her as affected, we remapped the disease to a 19.5-cM interval (DXS1219-DXS993) at Xp11.4-p21.1. This new interval overlapped both RP3 (RPGR) and COD1. Sequencing of the previously published exons of RPGR revealed no mutations, but a de novo insertion was detected in the new RPGR exon, ORF15. The identification of an RPGR mutation in a family with a severe form of cone and rod degeneration suggests that RPGR mutations may encompass a broader phenotypic spectrum than has previously been recognized in "typical" retinitis pigmentosa.