Quantification and In Silico Toxicity Assessment of Tazarotene and its Impurities for a Quality and Safe Drug Product Development.

Tazarotene is internationally accepted common name for ethyl 6-[(4,4-dimethylthiochroman-6-yl)ethynyl]nicotinate. It is a synthetic retinoid used for the topical treatment of mild to moderate plaque psoriasis, acne vulgaris and photo aging. To ensure the quality of drug product and drug substance, a LC-MS compatible UHPLC method was developed for quantification of drug and its related substances. Stationary phase with fused core particle technology is used for the separation of impurities. Limit of quantification and limit of detection of the method are 0.1 and 0.03%, respectively. Precision of the method for Tazarotene and all its related substances is less than 2.2% RSD. The correlation coefficient is >0.999. Accuracy of method is ranged from 95.3% to 107.0%. Application of this method in stability analysis has been demonstrated by analyzing stressed samples. Experimental design is used for the verification of robustness of the method. To ensure the safety, an in silico toxicity of the drug and its related substances were determined using TOPKAT and DEREK toxicity predictions Both UHPLC and in silico methods were validated as per the ICH Q2 and ICH M7 guidelines, which will enable a rapid product development of Tazarotene topical formulations while ensuring the safety and quality of product.

Quantification and In Silico Toxicity Assessment of Nimesulide and its Related Substances.

Availability of analytical method which is compatible with both Ultra high performance liquid chromatography (UHPLC) and Liquid chromatography mass spectrometry to identify and quantify the impurities in pharmaceutical products is an advantage for drug product development; such analytical method was developed for Nimesulide using a fused core column. Ammonium acetate and methanol were used as mobile phase in the gradient elution at a detection wavelength 230 nm. Quality-by-design principles helped in identifying the critical method development parameters and design space. Limit of detection and the limit of quantification are 0.025 and 0.075%, respectively. Precision of the method is <0.4% RSD and correlation coefficient is >0.999 for Nimesulide and all related substances. Accuracy of Nimesulide ranged from 99.3 to 100.4% at assay concentration. Application of this method in stability analysis has been demonstrated by analyzing stressed samples. The method was validated for specificity, linearity, accuracy, precision and robustness in compliance with International Conference on Harmonization (ICH) Q2(R1). This is the first reported UHPLC method for the estimation of Nimesulide and its related substances. According to ICH M7 guidelines, all impurities were assessed for genotoxicity, LD50, skin sensitization, irritation potential and carcinogenicity using Derek and TOPKAT software. Combined knowledge of analytical and toxicology assessment will help in developing safe and quality product.

Benign epithelioid peripheral nerve sheath tumour resembling schwannoma.

Peripheral nerve sheath tumours (PNST) with epithelial appearing cells compromise a heterogeneous group of neoplasms that are rare and diagnostically challenging. Of these, malignant PNSTs with epithelioid features (epithelioid MPNST) are commonly described in literature. However benign epithelioid PNSTs are rare and till date about 38 cases have been described in the literature. We report a benign epithelioid PNST with light microscopical and immunohistochemical features suggestive of schwannoma, presenting as a thigh mass in a 23-year-old female. The tumour was encapsulated, showed epithelioid cells in aggregates, and expressed vimentin and S-100 positivity. There was no expression of CD34, CK, EMA, CD99, p63 and HMB 45. Typical Antoni A and Antoni B areas were absent. At 18 months follow-up, the patient was well.

Inclusion complex of aprepitant with cyclodextrin: evaluation of physico-chemical and pharmacokinetic properties.

OBJECTIVE: Aprepitant (APR) is a water insoluble drug approved for the treatment of chemotherapy induced nausea and vomiting (CINV) and post-operative nausea and vomiting (PONV). The innovator Emend® is a formulation incorporating drug nanoparticles with good bioavailability (~67%). The objective of the current work was to evaluate the feasibility of formulating a cyclodextrin complex of APR with enhanced solubility/dissolution rate and concomitantly bioavailability. METHODS: The complex was prepared using two approaches: kneading and slurry method. The formulated complex was evaluated using DSC, XRPD and FT-IR studies. RESULTS: DSC, XRPD and FT-IR studies confirmed the interaction of ß-cyclodextrin with APR indicating formation of a true complex wherein the drug was encapsulated in the cyclodextrin cavity (inclusion phenomenon). In addition to inclusion complexation, non inclusion phenomenon viz., interaction among hydroxyl groups of cyclodextrin and APR was also observed. The saturation solubility and dissolution rate of drug complex was higher than that of aprepitant API. The rate (C(max)) and extent of absorption (AUC) of APR from the complex were found to be comparable to that of Emend® (Reference product). CONCLUSION: These studies established that cyclodextrin complexation may provide another viable and cost effective option for enhancing solubility and bioavailability of APR.

Liquid chromatography/microspray mass spectrometry for bacterial investigations.

Cellular proteins (biomarkers) specific to any individual microorganism, determined by the direct mass spectral analysis of the corresponding intact cellular suspension, can be applied for the rapid and specific identification of the organisms present in unknown samples. The components of the bacterial suspensions, after a rapid separation over a C18 reversed-phase microcapillary column, were directly subjected to on-line electrospray ionization followed by analysis using an ion trap tandem mass spectrometer. This approach is equally effective for gram-positive as well as gram-negative bacteria but has a distinct advantage over our earlier reported method involving matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). During electrospray ionitation mass spectrometry (ESI-MS), liquid samples can be directly analyzed and there is the potential for developing tandem mass spectral methods for more specific identification of the individual organisms present in crude bacterial mixtures. The total analysis time leading to unambiguous bacterial identification in samples was less than 10 minutes and the results were quite reproducible. Miniaturization of the instrumentation along with total automation of this simple process could have immense impact on field operations. Routine, rapid, cost-effective field monitoring of environmental samples, agricultural products, samples from food processing, industrial sites and health institutions for suspected bacterial contamination could be a reality in the near future. Potential utility in biological, medical, bioprocessing, pharmaceutical, and other industrial research is also enormous.

Mass spectrometric investigations on Conus peptides.

Molecular masses and primary structure determination of Conus peptides, such as alpha-, mu- and omega-conotoxins, conantokins and conopressins, were accurately measured by state-of-the-art mass spectrometric techniques using only 1-2 pmole quantities. Soft ionization of Conus peptides under electrospray, matrix-assisted laser desorption and continuous flow frit-FAB conditions produced their corresponding singly and multiply charged molecular ions which can be detected by mass spectrometric analysis. The molecular masses of Conus peptides were obtained by the deconvolution of the multiply charged pseudo-molecular ions. Mixture analysis without chromatographic separation can be accomplished by this approach. The ions formed during collision-induced dissociation of either singly or multiply charged ions of any reduced and derivatized peptide provided the corresponding sequences of the amino acids. Preliminary investigations indicate that the developed techniques and procedures could be applied in order to characterize the peptides present in unknown Conus venoms from the Bay of Bengal region.

Detection of pathogenic and non-pathogenic bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The proteins isolated from the whole cells of bacterial pathogens and related non-pathogenic simulants were analyzed directly, with minimal sample preparation by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Inspection of mass spectrometric profiles obtained from direct MALDI-MS analysis of the protein extracts revealed specific biomarkers for individual bacterial cells. The observed biomarkers enabled us not only to detect pathogenic bacteria (Bacillus anthracis, Yersinia pestis and Brucella meliteusis), but also to distinguish them from the corresponding non-pathogenic species. By examining a series of strains of several Bacillus species (anthracis, thuringiensis, cereus and subtilis), it was possible to derive genus, species and strain-specific biomarkers from the measured molecular masses of the intact proteins. Additional series of biomarkers were obtained from direct mass spectrometric analysis of tryptic digests of the protein extracts. The application of this technique for rapid chemotaxonomic classification of microorganisms is demonstrated.

Rapid identification of bacteria by direct matrix-assisted laser desorption/ionization mass spectrometric analysis of whole cells.

Several characteristic ions were observed during the direct analysis of a variety of both gram-negative and gram-positive intact bacterial cells by the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) technique. The entire process, involving absolutely no sample processing, could be completed in less than ten minutes. A number of specific biomarkers, generated reproducibly for each type of cell from the corresponding mass spectrum, permitted the identification, as well as the distinction, of pathogenic bacteria from their non-pathogenic counterparts. In addition, individual strains of a specific organism could also be differentiated easily. Some of these biomarkers correspond to those observed earlier during the MALDI-MS analysis of protein extracts of the same bacteria. This approach, which can yield valuable data for rapid classification and detection of microorganisms, represents a substantial breakthrough for rapid screening of environmental as well as biological samples.

Identification of disulfide bridges in a cardiotoxic peptide by electrospray ionization.

A new method was developed for subjecting a peptide to a specified number of Edman degradation cycles on an automated polypeptide sequencer and desorbing the residual peptide for further investigations. The procedure was applied in combination with electrospray ionization mass spectrometry to identify the four disulfide bridges present in a small tightly bound peptide. The task was accomplished using only a few nanomoles of the intact peptide.

Immunogenicity of a plasma-derived hepatitis B vaccine in children and adults.

Immunisation of health care workers and staff working in laboratory and hospital settings has been implemented since 1988. However due to the high cost of currently available HBV vaccine, many health personnel outside the Ministry of Health are not being immunised. This study sought to determine the immunogenicity of three doses of a low cost plasma-derived Korean HBV vaccine on employees of an institute for mentally handicapped and their spouses and children. We found that the Hepatitis B Vaccine-KGCC to be safe and immunogenic. The response to 10 mcg and 20 mcg Hepatitis B Vaccine-KGCC after third dose was good with 100% seroconversion.

Chirality in microcystins.

A new method has been developed to identify the isomers of amino acids by derivatization of the corresponding standards with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent or FDAA) and analysis of the diastereomeric derivatives by a liquid chromatography-thermospray mass spectrometry technique. Quantification of the FDAA derivatives that originate from standards was possible by using L-phenylalanine as the internal standard. The procedure was applied to determine the chiralities of the amino acids present in some previously uncharacterized blue-green algal peptides (microcystins).

Pharmacokinetics of two novel rectal controlled-release morphine formulations.

Administration of morphine by the oral route is not possible in cancer patients who are unable to swallow or are intolerant of oral morphine. Thus, there is a need for reliable alternate routes of drug administration. We compared the bioavailability of two prototype 30-mg morphine sulfate controlled-release suppository formulations (high and low viscosity) with 30-mg oral controlled-release morphine sulfate tablets in a 14-subject single-dose randomized, three-way crossover study. Venous blood samples were obtained immediately prior to and for 24 hr following each dose. Morphine concentrations were determined by radioimmunoassay. Compared with oral controlled-release morphine, the high- and low-viscosity suppository formulations had significantly greater bioavailability (AUC0-24 72.7 +/- 13.2 for the oral preparation versus 98.6 +/- 35.7 and 105.8 +/- 37.3 ng.hr/mL for the suppositories, respectively, P < 0.05), later peak concentration (tmax 2.3 +/- 0.8 versus 3.1 +/- 2.3 and 5.0 +/- 1.5 hr, respectively, P < 0.05), and longer half-value duration (4.3 +/- 1.6 versus 10.4 +/- 5.5 and 9.5 +/- 4.3 hr, respectively, P < 0.05). Peak concentration for the high-viscosity suppository formulation (Cmax 7.75 +/- 2.66 ng/mL) was significantly lower than the low-viscosity suppository (Cmax 9.23 +/- 2.85 ng/mL) and the oral tablet (Cmax 10.4 +/- 2.78 ng/mL) formulations (P < 0.05). The increased bioavailability observed with the two controlled-release suppositories may be the result of partial avoidance of hepatic biotransformation with rectal administration.

Mass spectral investigations on trichothecene mycotoxins. VIII--Thermospray ionization and collisionally activated dissociation of macrocyclic trichothecenes.

Several structurally similar, biologically active macrocyclic trichothecenes were ionized under thermospray conditions followed by collisionally activated dissociation of the ammonium adducts. Most of the observed daughter ions were formed by bond cleavage at the exocyclic ester bridges. Compounds with similar ester bridges formed several common daughter ions and underwent similar neutral losses. Fragmentation pathways proposed for the dissociation of the adducts were confirmed from the corresponding neutral loss spectra. Simple experiments designed for the sequential monitoring of the characteristic daughter ions were used for the rapid, direct and accurate analysis of macrocyclic trichothecenes in real samples. The primary drawback of the method is its inability to distinguish between isomers.

Mass spectral investigations on trichothecene mycotoxins. VII. Liquid chromatographic-thermospray mass spectrometric analysis of macrocyclic trichothecenes.

Thermally labile, polar toxic roridins and biologically active, isomeric baccharinoids were separated on a reversed-phase high-performance liquid chromatography column and effectively ionized under thermospray ionization conditions. The mass spectra indicated the formation of corresponding molecular ion-ammonium adducts in great abundance. Experiments designed for monitoring specific ions of these analytes at predesignated intervals were utilized for the accurate analysis of these macrocyclic trichothecenes in real, crude samples. A synthetically modified macrocyclic trichothecene, 8-ketoverrucarin A, was used as the internal standard for the detection and quantification of these compounds. Minimum detectable limits, during this first reported method for the unambiguous analysis of these structurally related macrocyclic trichothecenes, were determined to be 2-5 ng.

Structural characterization of toxic cyclic peptides from blue-green algae by tandem mass spectrometry.

Combined use of chemical degradation, derivatization, and tandem mass spectrometry for rapid structural characterization of toxic cyclic peptides from blue-green algae at the nanomole level is described. Previously, all blue-green algal toxins were thought to belong to a family of seven-residue cyclic peptides, having the general structure cyclo-D-Ala-L-Xaa-erythro-beta-methyl-D-isoaspartic acid-L-Yaa-Adda-D-isoglutamic acid-N-methyldehydroalanine, where Xaa and Yaa represent variable amino acids of the L configuration and Adda is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid. Structural characterization of two additional toxins indicates that further variability can exist within this family of naturally occurring toxic cyclic peptides. Isoaspartic acid and dehydroalanine can substitute for beta-methylisoaspartic acid and N-methyldehydroalanine, respectively.

Mass spectral investigations on trichothecene mycotoxins. V. Direct analysis of satratoxins by tandem mass spectrometric techniques.

Under chemical ionization conditions, satratoxins preferentially produce negatively charged molecular ions (M-) over the positively charged protonated molecules. Collisionally activated dissociation of the M-.ions resulted in daughter ions and neutral losses which were characteristic of the macrocyclic ester bridges. Direct tandem mass spectrometric methods of analyses wee developed and applied for the detection and quantification of satratoxins in Stachybotrys atra fermentation broths. Minimum detectable levels for satratoxin standards were 5 pg. A synthetically modified macrocyclic trichothecene, 8-ketoverrucarin A, was used as an internal standard for the quantification of satratoxins in fermentation samples.

Mass spectral investigations on trichothecene mycotoxins. II. Detection and quantitation of macrocyclic trichothecenes by gas chromatography/negative ion chemical ionization mass spectrometry.

A general, sensitive gas chromatographic/negative ion chemical ionization mass spectrometric (GC/NICIMS) method of analysis was developed for the detection and quantitation of several polar, thermally labile, toxic macrocyclic trichothecenes. The procedure involves the conversion of the molecules to their corresponding alcohols (verrucarols) by alkaline hydrolysis, followed by derivatization of the hydrolysate with heptafluorobutyrylimidazole and analysis by GC/MS technique under negative ion chemical ionization conditions. Nanogram (250 ng) quantities of several macrocyclic trichothecenes with different verrucarol and ester moieties were analyzed successfully with good precision by this procedure. The method was applicable for the accurate determination of at least low ppb levels of these macrocyclic trichothecenes in environmental samples, such as fungal products, fermentation broths, and plant samples. This is the first reported, well developed, sensitive, and applicable method for the detection and quantitation of these compounds in naturally occurring samples.