Immunogenicity and Induction of Functional Antibodies in Rabbits Immunized with a Trivalent Typhoid-Invasive Nontyphoidal Salmonella Glycoconjugate Formulation.

Typhoid fever due to Salmonella Typhi and invasive nontyphoidal Salmonella (iNTS) infections caused by serovars Enteritidis (SE) and Typhimurium (STm) are major pediatric health problems in sub-Saharan Africa. Typhoid has high complication rates, and iNTS infections have high case fatality rates; moreover, emerging antimicrobial resistance is diminishing treatment options. Vi capsule-based typhoid conjugate vaccine (Typbar-TCV™), licensed in India and pre-qualified by the World Health Organization, elicits durable immunity when administered to infants, but no iNTS vaccines are licensed or imminent. We have developed monovalent SE and STm glycoconjugate vaccines based on coupling lipopolysaccharide-derived core-O polysaccharide (COPS) to phase 1 flagellin protein (FliC) from the homologous serovar. Herein, we report the immunogenicity of multivalent formulations of iNTS COPS:FliC conjugates with Typbar-TCV™. Rabbits immunized with the trivalent typhoid-iNTS glycoconjugate vaccine generated high titers of serum IgG antibody to all three polysaccharide antigens for which anti-COPS IgG antibodies were directed primarily against serogroup-specific OPS epitopes. Responses to SE and STm FliC were lower relative to anti-COPS titers. Post-vaccination rabbit sera mediated bactericidal activity in-vitro, and protected mice after passive transfer against challenge with virulent SE or STm Malian blood isolates. These results support accelerated progression to clinical trials.

Development and validation of a stability-indicating assay including the isolation and characterization of degradation products of metaxalone by LC-MS.

A stability-indicating reverse-phase high-performance liquid chromatography-mass spectrometric method was developed and validated for the assay of metaxalone through forced degradation under acidic, alkaline, photo, oxidative and peroxide stress conditions. Separation of degradation products was accomplished on a reverse-phase Phenomenex C18 (250 × 4.6 mm, 5 µm) column thermostated at 25 °C using 10 mM aqueous ammonium acetate: methanol (35:65 v/v) as mobile phase in an isocratic mode of elution. The eluents were detected at 275 nm by photo diode array detector and mass detectors connected in series. Two unknown base hydrolysis products of metaxalone were identified and characterized as (a) methyl 3-(3,5-dimethylphenoxy)-2-hydroxypropylcarbamate and (b) 1-(3,5-dimethylphenoxy)-3-aminopropan-2-ol by MS, (1)H NMR and FTIR spectroscopy. The method was validated as per International Conference on Harmonization guidelines and metaxalone was selectively determined in presence of its degradation impurities, demonstrating its stability-indicating nature.

Evaluation of polysaccharide-based chiral stationary phases in quality control of (S)-mirtazapine.

High-performance liquid chromatographic methods were developed for separation of the enantiomers of mirtazapine and its four process-related substances. The direct separations were achieved on chiral stationary phases containing amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD-H), cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD-H) and cellulose tris(4-methylbenzoate) (Chiralcel OJ-H ). The experimental data were utilized to discuss the effects of the mobile phase composition, the nature of the alcoholic modifier and the specific structural features of the analytes on retention and separation. The elution sequence was determined under the optimized separation conditions.

Synthesis, structure-activity relationship of novel substituted 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylates as potential anti-mycobacterial and anticancer agents.

Series of 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives 7a-7zb, 8a-8d and 9a-9d were synthesized and screened for their in vitro anti-mycobacterial activity against Mycobacterium tuberculosis H(37)Rv (MTB) and cytotoxicity against three human cancer cell lines including A549, SK-N-SH and HeLa. The results indicate that six compounds are more potent and 7za is most effective anti-mycobacterial derivative compared to the standard drugs Ethambutol and Ciprofloxacin. However, 12 compounds exhibited cytotoxicity against human neuroblastoma cell line; amongst them the compound 7v is most effective compared to the standard drug Doxorubicin. This is the first report assigning in vitro anti-mycobacterial, anticancer and structure-activity relationship for this new class of 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-carboxylates.

High-performance liquid chromatographic determination of anti- hypertensive drugs on dried blood spots using a fluorescence detector--method development and validation.

A selective and sensitive high-performance liquid chromatography method with fluorescence detection for simultaneous determination of irbesartan, losartan and valsartan on dried blood spots (DBS) has been developed and validated. It involves solvent extraction of a punch of DBS followed by reversed-phase liquid chromatography on a Lichrospher(®) 100 RP-18e column. Fluorescence detection was performed at 259 and 385 nm as excitation and emission wavelengths, respectively. The detection limits of irbesartan, losartan and valsartan were 1.8, 3.6 and 1.8 ng/mL respectively. The mean recoveries of irbesartan, losartan and valsartan were 98.68, 98.42 and 97.81%, respectively. The mean inter-day and intra-day precisions of irbesartan, losartan and valsartan were 2.07 and 1.34%, 1.42 and 1.48%, and 3.20 and 2.15% respectively. The proposed method was simple and rapid. Design of experiments was used to evaluate the robustness of the method.

Development and validation of a RP-HPLC method for stability-indicating assay of gemifloxacin mesylate including identification of related substances by LC-ESI-MS/MS, 1H and 13C NMR spectroscopy.

A validated stability indicating RP-HPLC assay of gemifloxacin mesylate was developed by separating its related substances on an Inertsil-ODS3V-C18 (4.6 × 250 mm; 5 µm) column using 0.1% trifluoroaceticacid (pH 2.5) and methanol as a mobile phase in a gradient elution mode at a flow rate of 1.0 mL/min at 27°C. The column effluents were monitored by a photodiode array detector set at 287 nm. The method was validated in terms of accuracy, precision and linearity as per ICH guidelines. Forced degradation of gemifloxacin (GFX) was carried out under acidic, basic, thermal, photolysis and peroxide conditions and the degradation products were separated and characterized by ESI-MS/MS, (1) H and (13) C NMR spectroscopy. The method was successfully applied to the analysis of bulk drugs and the recoveries of gemifloxacin and impurities were in the range of 97.60-102.90 and 96.99-102.10%, respectively. No previous reports were found in the literature on identification of degradation products of gemifloxacin.

Rapid determination of rifaximin on dried blood spots by LC-ESI-MS.

The use of blood spot collection cards is a simple way to obtain specimens for therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in a clinical setting. A high-throughput liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) method for determination of rifaximin on dried blood spots (DBS) was developed and validated. It involves solvent extraction of a punch of DBS followed by reversed-phase LC on a monolithic column consisting of a silica rod with bimodal pore structure and detection by ESI-MS. Rifampicin was used as an internal standard (IS). The run time was within 5.0 min with a very low back-pressure at a flow rate of 0.5 mL/min. The assay was linear from 0.1 to 10 ng/mL. The mean recovery was 98.42%. The developed method is very simple, rapid and useful for clinical applications.

Reversed-phase liquid chromatographic determination of tramiprosate in rat plasma using evaporative light scattering detector.

A simple and reliable method was developed for determining blood concentrations of tramiprosate using reversed-phase HPLC with evaporative light scattering detection. The optimum evaporator and nebulizer temperatures for ELSD were set at 90 and 60°C respectively. The method was linear for a concentration range of 10-1000 µg/mL when 0.3 mL blood was used. The intra-assay precision ranged from 1.84 to 5.24%, and the coefficient of variation (CV) for a quality control sample at 10 µg/mL was 5.24% with a bias of -9.50% from the target value. The inter-assay precision ranged from 2.58 to 5.96%, and the CV for a quality control sample at 10 µg/mL was 5.96% with a bias of -7.50% from the target value. The method described here is suitable for management of tramiprosate in Alzheimer disease therapy.

Development of a validated LC method for enantiomeric separation and determination of adrafinil and its related substances on a Chiralcel OJ-H column connected to PDA and polarimetric detectors in series.

A rapid and reliable high-performance liquid chromatographic method for resolution of enantiomers of adrafinil [(±)-ADL], a novel vigilance promoting agent, and its synthetic intermediates was developed. The separation was carried out on a Chiralcel OJ-H using n-hexane-ethanol (62:38 v/v) as a mobile phase. The detection was carried out at 225 nm using a photodiode array (PDA) detector. The optical rotation and order of elution of enantiomers were assigned. The method is suitable not only for process development of ADL but also for quality assurance of bulk drugs and pharmaceuticals.

Development of a validated high-throughput LC-ESI-MS method for determination of sirolimus on dried blood spots.

A high-throughput liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) method for screening of sirolimus on dried blood spots (DBS) was developed and validated. It involves solvent extraction of a punch of DBS followed by reversed-phase LC on a relatively new monolithic column consisting of a silica rod with bimodal pore structure and detection by ESI-MS. The run time was less than 3 min with a very low backpressure at a flow rate of 0.5 mL/min. The method can analyze more than 100 samples in an 8 h working day, including sample preparation. The assay was linear from 1 to 100 ng/mL. The mean recovery was 92.42%. The mean inter-day and intra-day precisions were 1.23 and 1.41%, respectively. The developed method is simple, rapid and useful for clinical applications.

Enantioselective separation and determination of adrafinil and modafinil on Chiralcel OJ-H column in rat serum and urine using solid-phase extraction followed by HPLC.

A simple and rapid normal-phase HPLC method for enantiospecific separation of a psychostimulant, adrafinil (ADL), and its metabolite modafinil (MDL) in rat serum and urine was developed. The separation was accomplished on a normal-phase polysaccharide stationary phase Chiralcel OJ-H using n-hexane-ethanol (62:38 v/v) as a mobile phase at a flow rate of 1.0 mL/min. Detection was carried out at 225 nm using a photo diode array (PDA) detector. The elution order of the enantiomers was determined by a polarimeter connected in series with the PDA. ADL and its metabolite were recovered from rat serum and urine by solid phase extraction using Oasis HLB cartridges and the mean recoveries were >or=80%. The enantiomers were eluted within 15 min without any interference from endogenous substances. The calibration curves were linear (r(2) > 0.998) in the concentration range of 1.20-500 microg/mL for ADL and MDL. The assay was specific, accurate, precise and reproducible (intra- and inter-day precisions RSDs <7.2%). ADL in rat serum was stable over three freeze-thaw cycles at ambient temperature for 4 h. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.

Rapid determination of rifaximin in rat serum and urine by direct injection on to a shielded hydrophobic stationary phase by HPLC.

A simple and rapid reversed-phase HPLC method for determination of rifaximin in rat serum and urine was developed. Separation of rifaximin from biological matrix was achieved by direct injection of rat serum and urine onto a restricted-access medium, Supelco LC-Hisep, a shielded hydrophobic stationary phase, using acetonitrile:water:acetic acid (18:82:0.1 v/v/v) as a mobile phase. The linear range was 0.10-20 microg/mL (r(2 )> 0.999, n = 6), intraday and interday variation was <6.10%. The limits of detection and quantification were 0.03 (signal-to-noise ratio >3) and 0.10 microg/mL (signal-to-noise ratio >10), respectively. The method was successfully applied to pharmacokinetic studies of rifaximin after an oral administration to rats.

On-line 2D-LC-ESI/MS/MS determination of rifaximin in rat serum.

A highly sensitive and selective on-line two-dimensional reversed-phase liquid chromatography/electrospray ionization-tandem mass spectrometry (2D-LC-ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D-LC-ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C(18 )column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5-10 ng/mL (r(2) > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies.

Development and validation of a reversed-phase HPLC method for separation and simultaneous determination of process-related substances of mirtazapine in bulk drugs and formulations.

A simple and rapid reversed-phase high-performance liquid chromatographic method has been developed for the separation and simultaneous determination of related substances of mirtazapine in bulk drugs and pharmaceutical formulations. Six impurities, including one degradation product of mirtazapine, have been separated on a BDS Hypersil (4.6 x 250 mm; particle size 5 microm) column with a mobile phase consisting of 0.3% triethylamine (pH 3.0)-acetonitrile (78:22 v/v) eluted in an isocratic mode and monitored with a photo diode array detector at 215 nm. The chromatographic behavior of all the analytes was studied under variable compositions of different solvent systems, temperatures, buffer concentrations, and pH values. The method was validated in terms of accuracy, precision, and linearity. The inter- and intra-day assay precision was found to be < 0.98% [relative standard deviation; (RSD)] and the recoveries were in the range 95.54-102.22% with RSD < 2.21%. The correlation coefficients for calibration curves for mirtazapine as well as impurities were in the range of 0.9941-0.9999. The method was successfully applied to the analysis of commercial formulations and the recoveries of mirtazapine were in the range of 99.38-100.73% with < 0.52% RSD. The method is useful not only for rapid evaluation of the purity of mirtazapine, but also for the simultaneous determination of related substances in bulk drugs and pharmaceutical formulations.

LC-ESI-MS determination and pharmacokinetics of adrafinil in rats.

A highly sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for investigating the pharmacokinetics of adrafinil in rats was developed. Rat serum pretreated by solid-phase extraction (SPE) was analyzed by LC-MS/MS with an electrospray ionization (ESI) interface. The mobile phase consisted of acetonitrile:water:acetic acid (35:65:0.1, v/v/v) in an isocratic elution mode pumped at 1.0 ml/min. The analytical column (250 mm x 4.6 mm i.d.) was packed with Kromasil C(18) material (5.0 microm). The standard curve was linear from 16.5 to 5000 ng/ml. The assay was specific, accurate (R.S.D.<2.6%), precise and reproducible (within- and between-day precisions R.S.D. <7.0% and <9.0%, respectively). Adrafinil in rat serum was stable over three freeze-thaw cycles at ambient temperature for 6 h. The method had a lower limit of quantitation of 16.5 ng/ml, which offered high sensitivity for the determination of adrafinil in serum. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.

Continuous counter current extraction, isolation and determination of solanesol in Nicotiana tobacum L. by non-aqueous reversed phase high performance liquid chromatography.

A method of continuous counter current extraction in a large-scale production of solanesol from tobacco leaves was developed. The crude extract containing 15-20% solanesol was subjected to a series of steps, viz., saponification, solvent recrystallization and column chromatography. The pure material was characterized by FT-IR, ESI-MS, 1H NMR and 13C NMR spectrometry. The analysis was carried out by a simple and rapid non-aqueous reversed-phase high performance liquid chromatographic method on a Hypersil BDS C18 column (250 mm x 4.6mm, particle size 5 microm) with isopropyl alcohol-methanol (60:40, v/v) as mobile phase and detection at 215 nm. The product purity was between 95 and 98% (w/w) as determined by HPLC.

Use of solid-phase extraction, reverse osmosis and vacuum distillation for recovery of aromatic sulfonic acids from aquatic environment followed by their determination using liquid chromatography-electrospray ionization tandem mass spectrometry.

Three different sample preparation techniques (i) solid-phase extraction, (ii) reverse osmosis and (iii) vacuum distillation have been investigated and the recoveries were compared for determination of highly water-soluble benzene and stilbene sulfonic acids in aqueous environment by liquid chromatography with photodiode array (PDA) and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The recoveries were quite high using vacuum distillation (>90%) compared to solid phase extraction and reverse osmosis. The negative ion ESI mass spectra containing the peaks of quasimolecular ion [M-H]- allow the molecular mass determination of unknown compounds whereas the structures were proposed using fragments obtained from MS/MS analysis of [M-H]- ions. At lower fragmentation voltages only the quasimolecular ion [M-H]- was observed and as fragmentation voltages increased, it led to the formation of fragment ions corresponding to [M-H-SO3]-, [M-H-SO2]-, and SO3-. The detection limits were 1-28 microg/L with LC-ESI-MS. The sample collected from wastewater treatment plant was found to contain 21.1, 13.3, 12.1, 41.8 and 9.9 microg/L of cis-4,4(l)-diaminostilbene-2,2(l)-disulfonic acid (cis-DASDA), trans-4,4(l)-diaminostilbene-2,2(l)-disulfonic acid (trans-DASDA), 3-amino acetanilide-4-sulfonic acid (3-AASA), 4-chloroaniline-2-sulfonic acid (4-CASA), 2-chloroaniline-5-sulfonic acid (2-CASA), respectively.

Rapid separation and determination of process-related substances of paracetamol using reversed-phase HPLC with photo diode array as a detector.

A simple and rapid gradient reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of paracetamol and its related compounds in bulk drugs and pharmaceutical formulations has been developed. As many as nine process impurities and one degradation product of paracetamol have been separated on a Symmetry C18 column (4.6 x 250 mm i.d., particle size 5 microm) with gradient elution using 0.01 M potassium dihydrogen phosphate buffer (pH 3.0) and acetonitrile as mobile phase and photo diode array detection at 215 nm. The chromatographic behavior of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH values. The correlation coefficients for calibration curves for paracetamol as well as impurities were in the range of 0.9951 - 0.9994. The proposed RP-LC method was successfully applied to the analysis of commercial formulations; the recoveries of paracetamol were in the range of 99-101%. The method could be of use not only for rapid and routine evaluation of the quality of paracetamol in bulk drug manufacturing units but also for detection of its impurities in pharmaceutical formulations.

Liquid-chromatographic separation and determination of process-related impurities, including a regio-specific isomer of celecoxib on reversed-phase C18 column dynamically coated with hexamethyldisilazane.

A simple and rapid reversed-phase high-performance liquid-chromatographic method for the separation and determination of process-related impurities of celecoxib (CXB) in bulk drugs and pharmaceuticals was developed. The separation of impurities viz., 4-methylacetophenone (I), 1-(4-methylphenyl)-4,4,4-trifluorobutane-1,3-dione (II), 4-hydrazinobenzene sulfonamide (III) and a regio-specific isomer [3-(4-methylphenyl)-5-trifluoromethyl-1H-pyrazole-1-yl]-benzenesulfonamide (IV), was accomplished on an Inertsil ODS-3 column dynamically coated with 0.1% hexamethyldisilazane (HMDS) in acetonitrile:water (55:45 v/v) as a mobile phase and detection at 242 nm using PDA at ambient temperature. The chromatographic conditions were optimized by studying the effects of HMDS, an organic modifier, time of silanization and column temperature. The method was validated and found to be suitable not only for monitoring the synthetic reactions, but also to evaluate the quality of CXB.

Preparative isolation and characterization of some minor impurities of astaxanthin by high-performance liquid chromatography.

A simple and rapid preparative high-performance liquid chromatography (HPLC) method has been developed to isolate and characterize some minor impurities of astaxanthin using a normal-phase Lichrosorb silica column with n-hexane-acetone-tetrahydrofuran (90:2:8, v/v/v) as mobile-phase and detection at 475 nm. The isolated impurities were characterized as astacene, dehydro astacene and apoastaxanthinal by UV-vis, ESI-MS, 1H and 13C NMR spectroscopy and the molecular structures were assigned. The impurities collected using the developed conditions were over 98% pure.