Development of a validated LC-MS/MS method for determination of doxofylline on rat dried blood spots and urine: application to pharmacokinetics.

A rapid and highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determination of doxofylline on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse phase C18 column (250 mm × 4.6 mm, 5 µm), using 20 mM ammonium acetate (pH adjusted to 3.5 with trifluoroacetic acid) and acetonitrile (75:25 v/v) as a mobile phase at 25 °C. LC-MS detection was performed with selective ion monitoring using target ions at m/z 267 and m/z 195 for doxofylline and caffeine used as internal standard respectively. The calibration curve showed a good linearity in the concentration range of 1-5000 ng/mL. The effect of hematocrit on extraction of doxofylline from DBS was evaluated. The mean recoveries of doxofylline from DBS and urine were 93.46 and 89.86% respectively. The intra and inter-day precisions were less than 4.28% in DBS as well as urine. The limit of detection and quantification were 0.24 and 0.84 ng/mL in DBS and 0.28 and 1.00 ng/mL in urine samples respectively. The method was validated as per ICH guidelines and successfully applied to a pharmacokinetic study of doxofylline in rats.

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.

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 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.

Evaluation of antimicrobial, antioxidant and wound-healing potentials of Holoptelea integrifolia.

The methanolic extracts of Holoptelea integrifolia (Roxb.) (Urticaceae) leaves (MLE) and stem bark (MSBE) were studied for the wound-healing potential. Since wound healing is severely hampered by microbial infection and reactive oxygen species (ROS), this study was undertaken to evaluate antimicrobial and antioxidant activity apart from wound-healing activity. The antimicrobial property of the Holoptelea was studied against the six bacterial and five fungal strains using the agar well diffusion method and minimum microbicidal concentration and minimum inhibitory concentration were determined for each strain, in which methanolic extract of stem bark (MSBE) has shown bigger zone of inhibition (11.3-20.4 mm) than methanolic extract of leaves (MLE) (9.6-14.9 mm). The anti-oxidant activity was evaluated by DPPH free radical scavenging activity using HPLC method. The IC(50) values obtained for MSBE (TPC: 78.53+/-1.26 mg/g) and MLE (TPC: 57.71+/-1.45 mg/g) were 37.66+/-0.48 and 50.36+/-0.59 microg/well, respectively. In excision wound model, more than 90% wound healing was recorded in treated groups by 14 days of post surgery, where as only 62.99% was observed in the control group. In incision model, higher breaking strengths and higher hydroxyproline content in treated groups suggested higher collagen re-deposition than the control group. Finally, histopathology studies conformed wound-healing activity of Holoptelea integrifolia.