Supported liquid extraction and LC-MS-MS determination of iloperidone and olanzapine in rat plasma: Application to a pharmacokinetic study.

A sensitive, selective rapid bioanalytical assay method was developed and quantification of iloperidone (ILP) and olanzapine (OLZ) in rat plasma was done by mass spectrometry. Systematic sample preparation and extraction procedure were carried out by supported liquid extraction using dichloromethane to extract both the eluents (ILP and OLZ) from rat plasma samples. The extorted samples were injected on a selective Waters XTerra® C18 reverse-phase bonded column (250 × 4.6 mm i.d., 5 µm) using acetonitrile and 15 mM ammonium formate containing 0.05% trifluoroacetic acid (60:40 v/v) for isocratic elution mode and detected by mass spectrometry. Calibration curves were drawn with the respective assay statistical data and showed linear regression coefficients greater than 0.9996 over the concentration ranges 2-5,000 ng/mL for ILP and OLZ, respectively. The absolute mean recoveries were found to be in the replicate range of 87.12-94.47%, respectively. The obtained results by the method revealed good intra and interday assay performance in terms of 1.70-5.90% precision and 0-5% accuracy. The validated bioassay method has been successfully applied to the pharmacokinetics in rats.

Identification of Drug Targets in Helicobacter pylori by in silico Analysis: Possible Therapeutic Implications for Gastric cancer.

Helicobacter pylori colonize stomach, inducing gastritis, ulcers and gastric cancer. Drugs are used to relieve pain, but not H. pylori infections. Hence, there is a need for discovery of drug targets and drugs for H. pylori. An objective of this current study is to identify drug targets for H. pylori. RAST was used to compare genomes of 23 H. pylori strains with Homo sapiens sapiens, other Helicobacter species (H. acinonychis, H. hepaticus, H. mustalae) and among them, to identify 13471 unique genes. Bacterial genes which are non-homologous to humans and essential for pathogen are identified using BLASTp. Later, 29 potential drug targets were identified by subjecting these genes to property analysis. Eleven of the 29 drug targets are already experimentally validated, lending credence to our approach. These methods have enabled rapid identification of drug targets with possible therapeutic implications for gastric cancer.

Thermodynamic evaluation of immobilized cellulose tris(3,5-dichlorophenylcarbamate) as a stationary phase for liquid chromatographic separation of darunavir enantiomers.

Liquid chromatographic separation of darunavir (DRV) enantiomers was studied on Chiralpak IC column containing immobilized cellulose tris(3,5-dichlorophenylcarbamate) using a variety of mobile phase solvents at different temperatures. The separations were accomplished under normal phase conditions using different compositions of n-hexane, organic modifier (2-propanol, ethanol or 1-propanol) and diethyl amine (0.1%) as mobile phase solvents. The effect of volume and nature of organic modifier and column temperature on retention, separation and resolution were studied. Van't Hoff plots (ln k' vs 1/T) were drawn from the chromatographic retention data to calculate to apparent thermodynamic parameters and explain the interactions between the DRV enantiomers and cellulose tris(3,5-dichlorophenylcarbamate) immobilized on silica.

Stereo-specific LC and LC-MS bioassays of antidepressants and psychotics.

Stereochemistry in drug action is gaining importance because the enantiomers often differ in their biological activity and pharmacokinetic profiles. The use of racemic drugs may contribute to adverse effects owing to the presence of either inactive or toxic enantiomers. Most of the drugs currently used to treat psychiatric disorders, including depression, contain one or more chiral centers and are mostly sold as racemates. Single-enantiomer drugs provide greater selectivity for their biological targets, improved therapeutic indices and better pharmacokinetics compared with racemates. Therefore it is of great importance to monitor body fluid/tissue levels of drugs used to treat depression and psychiatric disorders. The present manuscript gives an overview of liquid chromatographic and mass spectrometric techniques reported during 2000-2013 for enantiomeric separation of various classes of antidepressants, viz. selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, noradrenergic and specific serotonergic antidepressants, norepinephrine reuptake inhibitors, norepinephrine-dopamine reuptake inhibitors, nonamphetamine central nervous system stimulants, serotonin and dopamine inhibitors and γ-aminobutyric acid receptor agonists in biological matrices. Techniques used for extraction, separation and quantification are discussed.

LC-Q-TOF-MS/MS determination of darunavir and its metabolites in rat serum and urine: application to pharmacokinetics.

A simple, rapid and reliable liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC-Q-TOF-MS/MS) method was developed and validated for simultaneous determination of darunavir and its metabolites in rat serum and urine. The separation was accomplished on an Agilent RP-18 (250×4.6mm, 5µm) column using 20mM ammonium acetate and methanol (40:60, v/v) as a mobile phase at a flow rate of 1.0mL/min in an isocratic mode. The [M+H](+) ions of darunavir (m/z 548) and metabolites-I (m/z 392) were monitored in positive mode of ionization, while [M-H](-) ion of metabolite-II (m/z 172) in negative mode selectively. The matrix effects of rat serum and urine were found to be negligible and the recoveries were 87-93% for all the analytes. The short and long term stability of darunavir and its metabolites was within acceptable limits and the lower limits of quantification were in the range of 3.63-5.24ng/mL with a linear range of 5-5000ng/mL in rat serum as well as urine. The method exhibited good intra- and inter-day performance in terms of 2.54-8.92% precision and 0-5% accuracy. The method was successfully applied to a single-dose pharmacokinetic study of darunavir boosted with ritonavir in Wistar rats.

Precolumn o-phthalaldehyde-N-acetyl-L-cysteine derivatization followed by RP-HPLC separation and fluorescence detection of sitagliptin enantiomers in rat plasma.

An indirect reversed-phase high-performance liquid chromatographic separation and fluorescence detection of sitagliptin enantiomers in rat plasma was developed and validated. Deproteinized rat plasma containing racemic sitagliptin was derivatized with o-phthalaldehyde and N-acetyl-L-cysteine under alkaline conditions, converted to diastereomers, and separated on a Lichrospher 100 RP-18e column using 20 mM phosphate buffer and methanol (45:55 v/v) as a mobile phase under isocratic mode of elution at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 330 and 450 nm as excitation and emission wavelengths, respectively. The method was linear in the range of 50-5000 ng/ mL for both enantiomers. The intra- and interday accuracy and precision were within the predefined limits of ≤15% at all concentrations. The method was successfully applied to a pharmacokinetic study of sitagliptin after 5 mg/kg oral administration to Wistar rats. Robustness of the method was evaluated using design of experiments.

Enantioselective separation and simultaneous determination of tolperisone and eperisone in rat plasma by LC-MS/MS.

Tolperisone and eperisone used as muscle relaxants possess one chiral center each and exist as two optical isomers for each drug. Therefore, enantioselective assays to measure each enantiomer in biological matrices are of great importance. In the present study a simple and complete reverse-phase liquid chromatography tandem mass spectrometric method for separation and enantioselective determination of tolperisone and eperisone in rat plasma was developed. The analytes were extracted from rat plasma by a simple protein precipitation method with acetonitrile as the extraction solvent. The enantioselective separation of analytes was achieved on a Cellulose Tris (4-chloro-3-methylphenylcarbamate) chiral column with a mobile phase of acetonitrile: 10 mM ammonium acetate in an isocratic mode of elution and mass spectrometric detection. The calibration curve for each enantiomer was found to be linear over 0.2 to 20 ng/mL for each enantiomer. The proposed method exhibited good intra- and interday precision (% CV) ranged between 0.95-6.05% and 1.11-8.21%, respectively. The intra- and interday accuracy for the proposed assay method ranged between 94.0-100.5% and 92.7-102.1%, respectively. The proposed method was validated as per regulatory guidelines.

Determination of gemifloxacin on dried blood spots by hydrophilic interaction liquid chromatography with fluorescence detector: application to pharmacokinetics in rats.

A highly selective, sensitive and rapid hydrophilic liquid interaction chromatographic method was developed and validated for determination of gemifloxacin on dried blood spots. The chromatographic separation was achieved on a reversed-phase zwitterionic hydrophilic interaction liquid chromatographic ZIC®HILIC-C18 (4.6 × 100 mm; 5 µm) column using acetonitrile-10 mM ammonium acetate (pH 3.5; 80:20, v/v) as a mobile phase in an isocratic elution mode at a flow rate 0.6 mL/min at 27 °C. An on-line fluorescence detector set at excitation and emission wavelengths of 269 and 393 nm, respectively was used for monitoring column eluents. Ciprofloxacin was used as an internal standard. The method was validated for accuracy, precision, linearity and selectivity by design of experiments following ICH guidelines. The assay exhibited a linear range of 25-5000 ng/mL for gemifloxacin on dried blood spots. The lower limit of detection was found to be 10 ng/mL. The intra- and inter-assay coefficients of variation did not exceed 7.4% deviation of the nominal concentration. The recovery of GFX from dried blood spots was >95.0% and its stability was excellent with no evidence of degradation during sample processing for at least 3 months storage in a freezer at -20 °C.

LC-ESI-MS/MS determination of in vivo metabolites of almotriptan in rat plasma, urine and feces: application to pharmacokinetics.

A highly sensitive and specific liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method for investigating the in vivo metabolites of almotriptan in rat plasma, feces and urine was developed. Chromatographic separation was achieved on a Lichrospher RP-18 column (250 mm × 4.6 mm, 5 µm), using 20mM ammonium acetate (pH 3.5) and acetonitrile (60:40, v/v) as a mobile phase at 25°C. MS/MS detection was performed by positive ion electrospray ionization using target ions at m/z 336 [M+H]⁺, m/z 368 and m/z 282 [M+H]⁺ for almotriptan and its two metabolites, respectively. Two metabolites viz., γ-aminobutyric acid and sulfonamide were detected in plasma as well as feces after 24 h of oral administration of almotriptan, while only γ-aminobutyric acid was found in urine. The method was sensitive with a lower limit of quantification of 1.43 ng/mL and linear over the range of 1.43-5000 ng/mL in plasma. The method was validated and successfully applied to a pharmacokinetic study of almotriptan in rat plasma using sumatriptan as an internal standard. The peak plasma concentration (C(max)) after 0.3h of 5mg/kg oral dose of almotriptan was determined to be 69.85 ng/mL.

LC-ESI-MS/MS determination of paclitaxel on dried blood spots.

A simple and rapid high-performance liquid chromatography-tandem mass spectrometric assay for determination of paclitaxel on rat dried blood spots was developed and validated. The extracted sample was chromatographed without further treatment using a reverse-phase Oyster ODS3, 4.6 × 50 mm, 3 µm column with mass spectrometry detection. The mobile phase comprised of acetonitrile-water, 60:40 v/v, with a flow rate of 0.4 mL/min was used. The calibration was linear over the range 0.2-20 ng/mL. The limits of detection and quantification were 0.08 and 0.2 ng/mL, respectively. The intra- and inter-day precision (CV%) and accuracy (relative error %) were less than 10 and 12%, respectively.

Two-dimensional LC-MS/MS determination of antiretroviral drugs in rat serum and urine.

A simple, rapid, reliable and highly sensitive on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometric (2D-LC/MS/MS) method to determine antiretroviral drugs viz., abacavir (ABC), nevirapine (NVP) and indinavir (IDV) in rat serum and urine was developed and validated. The analytes were extracted on-line from rat serum and urine by a restricted access material (RAM) column and back-flushed into the reversed-phase C18 column for separation by LC. Detection was carried out by ESI-MS/MS. The developed method showed good selectivity, accuracy and precision for quantification of the antiretroviral drugs in rat serum and urine. Quantification limits for abacavir and nevirapine were 4.0 ng ml(-1), whereas for indinavir 4.7 ng ml(-1). The calibration graphs were linear in the range of 4-50 ng ml(-1)for abacavir, nevirapine and indinavir. The method was successfully applied to study the pharmacokinetics of antiretroviral in rats.

Simultaneous separation and determination of coenzyme Q(10) and its process related impurities by NARP-HPLC and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS).

A non-aqueous reversed phase high performance liquid chromatographic (NARP-HPLC) method for determination of coenzyme Q(10) in pharmaceutical preparations has been developed using Kromosil C(8) column with acetonitrile and isopropyl alcohol (84:16, v/v) as a mobile phase. Photodiode array (PDA) detector set at 210 nm was used for monitoring of the eluents. The method is simple, rapid, selective and capable of separating all process impurities at trace level with detection limits <0.1 microg/ml. It has been validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The linearity range was 50-300 microg/ml. The percentage recoveries ranged from 95.10 to 101.02. The method was found to be suitable not only for monitoring the reactions during the process development but also quality assurance of coenzyme Q(10). For identification of related substances atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS) was used.

Determination of antibiotics in aquatic environment by solid-phase extraction followed by liquid chromatography-electrospray ionization mass spectrometry.

A robust and sensitive solid-phase extraction followed by liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) method for determination of antibiotics viz., fluoroquinolones, sulfamethoxazole, trimethoprim and cephalosporines in surface waters was developed. The sample recoveries on Oasis HLB cartridges were found to be >80%. Identification was carried out by LC-ESI-MS/MS. The positive ion ESI mass spectra containing the peaks of quasimolecular ions [M+H](+) allowed the determination of molecular masses whereas the fragment ions obtained by MS/MS of [M+H](+) ions permitted the structural assignment. Quantification was carried out by selective ion monitoring (SIM) using the quasimolecular ions [M+H](+) of the parent compounds. The detection and quantification limits were found to be in the range of 0.6-8.1 and 2.0-24.0 microg/L. The surface waters of different lakes and tanks of Hyderabad, India were found to contain a few antibiotics.

Isolation and characterization of process related impurities and degradation products of bicalutamide and development of RP-HPLC method for impurity profile study.

A reversed-phase high-performance liquid chromatographic method was developed for determination of process impurities and degradation products of bicalutamide in bulk drug and pharmaceutical formulations. The separation was accomplished on a Symmetry C(18) (4.6 mm x 250 mm; particle size 5 microm) column under isocratic mode. The mobile phase was 0.01 M KH(2)PO(4) (pH 3.0):acetonitrile (50:50 v/v) and a PDA detector set at 215 nm was used for detection. Forced degradation of bicalutamide was carried out under thermal, photo, acidic, alkaline and peroxide conditions. The unknown process impurities and alkaline degradation products were isolated and characterized by ESI-MS/MS, (1)H NMR and FT-IR spectral data. Under alkaline conditions bicalutamide was degraded in to an acid and an amine. The kinetics of degradation was studied. The proposed method was validated and successfully applied to the analysis of commercial formulations. Thus, the developed method can be used for process development as well as quality assurance of bicalutamide in bulk drug and pharmaceutical formulations.

Development of a validated RP-LC/ESI-MS-MS method for separation, identification and determination of related substances of tamsulosin in bulk drugs and formulations.

A reversed-phase high performance liquid chromatographic (RP-HPLC) method for evaluation of purity of tamsulosin in bulk drugs and pharmaceuticals was developed. The separation was accomplished on an Inertsil C(18) column using 10 mM ammonium acetate: acetonitrile as a mobile phase in a gradient elution mode. A photodiode array detector set at 280 nm was used for detection. The impurities were identified by ESI-MS-MS. The detection limits were 0.06-0.11 microg/ml. The method was validated with respect to accuracy, precision, linearity, ruggedness and limits of detection and quantification. It finds application not only for monitoring the reactions during the process development but also on quality assurance of tamsulosin.

Separation, characterization, and quantitation of process-related substances of the anti-hypertensive drug doxazosin mesylate by reversed-phase LC with PDA and ESI-MS as detectors.

A simple and rapid reversed-phase liquid chromatography (LC) method with photodiode array (PDA) and electrospray ionization (ESI)-mass spectrometry (MS) as detectors was developed and validated to separate, identify, and quantitate the related substances of Doxazosin mesylate (DXZN) for monitoring the reactions involved during process development. The high-performance liquid chromatography profiles of related-substances of DXZN are used as fingerprints to follow the procedures used in the manufacturing units. The separation is accomplished on an Inertsil ODS-3 column with acetonitrile-ammonium acetate (10 mM, pH 4.0) as the mobile phase, using a gradient elution mode and monitoring the eluents by a photodiode array detector at 265 nm at ambient temperature. LC-ESI-MS-MS is used to identify the additional impurities formed during the synthesis. The identified impurities were synthesized and characterized by UV, Fourier transform-IR, 1H NMR, and MS data. The detection limits for the impurities are 0.74 - 4.14 x 10(-9) g, and the method is found to be suitable not only for the monitoring of synthetic reactions, but also for quality assurance of DXZN in bulk drugs and formulations.

Enantiospecific assay of citadiol--a key intermediate of escitalopram by liquid chromatography on Chiralpak AD-H column connected with UV and polarimetric detectors in series.

A simple, rapid, selective and reproducible LC method for separation and quantitative determination of citadiol (CTD), a key intermediate of escitalopram has been developed. An optimum resolution > 3.0 was achieved on Chiralpak AD-H (250 mm x 4.6 mm); 5 microm column connected with UV and polarimetric detectors in series. The effects of organic modifiers, viz., methanol, ethanol, n-propanol and 2-propanol on enantioselectivity were evaluated. The limits of detection (LOD) and quantification (LOQ) were 0.02 microg/ml, 0.03 microg/ml and 0.07 microg/ml, 0.10 microg/ml for R-CTD and S-CTD enantiomers, respectively. The linearity of the method was studied in the range of 0.07-300 microg/ml and 0.1-300 microg/ml for R-CTD and S-CTD, respectively and the r2 was > or = 0.9999. The inter- and intra-day assay precision was less than 0.74% (%R.S.D.) and the recoveries were in the range 99.68-100.72% with %R.S.D. < 0.49%.

An overview of recent applications of inductively coupled plasma-mass spectrometry (ICP-MS) in determination of inorganic impurities in drugs and pharmaceuticals.

The recent applications of inductively coupled plasma-mass spectrometry (ICP-MS) in determination of trace level inorganic impurities in drugs and pharmaceuticals have been reviewed. ICP-MS coupled with LC, GC and CE was used for speciation of heavy metals in pharmaceutical products. The review covers the period from 1995 to 2005 during which the technique was applied not only for determination of metallic impurities but also the assay of various trace elements in pharmaceuticals.

Development and validation of a reversed phase liquid chromatographic method for separation and determination of related-substances of modafinil in bulk drugs.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method for determination and evaluation of purity of modafinil in bulk drugs using Kromasil C(18) column with acetonitrile: 0.02M ammonium acetate as a mobile phase in gradient elution mode at 30 degrees C and detection at 225nm using photodiode array detector has been developed. The effects of pH, temperature and the percent of organic modifier on resolution were studied. Related substances, viz, sulphide, sulphoxide, sulphones of the modafinil, acid and ester derivatives, were separated and quantified. The method was found to be simple, rapid, selective and capable of detecting all process related impurities at trace levels in the finished products of modafinil with detection limits of 0.6-2.4x10(-8)g. The method was validated with respect to accuracy, precision, linearity, ruggedness, and limits of detection and quantification. It was found to be suitable not only for monitoring the reactions during the process development but also quality assurance of modafinil.

Sublethal effects of profenofos on locomotor behavior and gill architecture of the mosquito fish, Gambusia affinis.

Subacute studies of profenofos on mosquito fish, Gambusia affinis, were carried out for 20 days to assess the locomotor behavior and structural integrity of gill in relation to bioaccumulation and targeted enzyme acetylcholinesterase (AChE; EC 3.1.1.7). The sublethal concentration of 0.13 mg/L (1/5 of LC50) altered locomotor behavior such as distance traveled and swimming speed in exposed fish. This could be due to inhibition in the activity of acetylcholinesterase and deformities in the primary and secondary lamella of gill. The bioaccumulation values indicated that the accumulation of profenofos was highest in viscera followed by head and body. The average bioconcentration factor values are 254.83, 6.18, and 2.52 microg/g for viscera, head, and body. The findings revealed that profenofos is highly toxic even at sublethal concentrations to the mosquito fish, Gambusia affinis.