Khaled H. Assi

Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.

Aerodynamic characteristics of a dry powder inhaler at low inhalation flows using a mixing inlet with an Andersen Cascade Impactor.

The aerodynamic characteristics of the dose emitted from a dry powder inhaler (DPI) are inhalation flow dependent but have not been determined for low flows. We have designed novel methodology to measure these at <28.3 lmin(-1). The original Andersen Cascade Impactor (ACI) designed for use at 60 lmin(-1) was adapted to include a mixing inlet (MIXINLET) which allows inhalation flows through the DPI from 5 to 60 lmin(-1). The mean fine particle dose (FPD) from a formoterol Turbuhaler using the MIXINLET method at 10, 20, 28.3, 40 and 60 lmin(-1) was 0.55, 1.39, 1.80, 2.88 and 5.86 microg and the mass median aerodynamic diameter (MMAD) was 6.6, 6.0, 5.4, 5.1 and 2.8 microm. Similarly, the FPD using the ACI method was 0.13, 0.69, 1.50, 2.48 and 5.42 microg and MMADs were 12.2, 7.4, 5.5, 4.8 and 2.7 microm. The accuracy of the original ACI <28.3 lmin(-1) is unknown. The ACI with the mixing inlet allows the determination of the in vitro dose emission properties of DPIs at flows <28.3 lmin(-1) whilst maintaining a constant flow through the ACI. This methodology, therefore, can help to focus attention to the lowest inhalation flow required for a DPI.

Microemulsion high performance liquid chromatography (MELC) method for the determination of terbutaline in pharmaceutical preparation.

A robust and sensitive microemulsion HPLC (MELC) method using oil-in-water microemulsion mobile phase was developed and used for the determination of terbutaline in Bricanyl(®) Turbuhaler. The applicability of microemulsion as an eluent for reversed phase HPLC was examined. In addition, the effect of operating parameters on the separation behaviour was studied. The samples were injected into C18 Spherisorb (250mm×4.6mm×5μm) columns at 25°C using a flow rate of 1ml/min. The mobile phase was 95.5% aqueous orthophosphate buffer (adjusted to pH 3 with orthophosphoric acid), 0.5% ethyl acetate, 1.5% Brij35, and 2.5% 1-butanol, all w/w. The terbutaline peak was detected by fluorescence, using excitation and emission wavelengths of 267 and 313nm, respectively. The accuracy of method was >99% and the calibration curve was linear (r(2)=0.99). The limit of detection (LOD) and limit of quantitation (LOQ) were 8μg/L and 26μg/L, respectively. The intra-day and inter-day precisions (in term of % coefficient of variation) were<1.46% and <0.97%, respectively. The influence of the composition of the microemulsion system was also studied and the method was found to be robust with respect to some changes of the microemulsion components. The microemulsion HPLC method has been applied to determine the content of the emitted dose and the fine particle dose of terbutaline in a Bricanyl(®) Turbuhaler.

Dose emission and aerodynamic characterization of the terbutaline sulphate dose emitted from a Turbuhaler at low inhalation flow.

Previously, dose emission below 30 L min−1 through DPI has not been routinely determined. However, during routine use some patients do not achieve 30 L min−1 inhalation flows. Hence, the aim of the present study was to determine dose emission characteristics for low inhalation flows from terbutaline sulphate Turbuhaler. Total emitted dose (TED), fine particle dose (FPD) and mass median aerodynamic diameter (MMAD) of terbutaline sulphate Turbuhaler were determined using inhalation flows of 10–60 L min−1 and inhaled volume of 4 L. TED and FPD increase significantly with the increase of inhalation flows (p <0.05). Flows had more pronounced effect on FPD than TED, thus, faster inhalation increases respirable amount more than it increases emitted dose. MMAD increases with decrease of inhalation flow until flow of 20L min−1 then it decreases. In vitro flow dependent dose emission has been demonstrated previously for Turbuhaler for flow rates above 30 L min−1 but is more pronounced below this flow. Minimal FPD below 30 L min−1 suggests that during routine use at this flow rate most of emitted dose will impact in mouth. Flow dependent dose emission results suggest that Pharmacopoeias should consider the use variety of inhalation flows rather than one that is equivalent to pressure drop of 4 KPa.

Relative bioavailability of terbutaline to the lung following inhalation, using urinary excretion

AIMS The aim of the study was to determine the relative lung and systemic bioavailability of terbutaline. METHODS On separate days healthy volunteers received 500 µg terbutaline study doses either inhaled from a metered dose inhaler or swallowed as a solution with and without oral charcoal. Urine samples were provided at timed intervals post dosing. RESULTS Mean (SD) urinary terbutaline 0.5 h post inhalation, in 12 volunteers, with (IC) and without (I) oral charcoal and oral (O) dosing was 7.4 (2.2), 6.5 (2.1) and 0.2 (0.2) µg. I and IC were similar and both significantly greater than O (P<0.001). Urinary 24 h terbutaline post I was similar to IC + O. The method was linear and reproducible, similar to that of the urinary salbutamol method. CONCLUSIONS The urinary salbutamol pharmacokinetic method post inhalation applies to terbutaline. Terbutaline study doses can replace routine salbutamol during these studies when patients are studied.

Smart nanocrystals of artemether: fabrication, characterization, and comparative in vitro and in vivo antimalarial evaluation

Artemether (ARTM) is a very effective antimalarial drug with poor solubility and consequently low bioavailability. Smart nanocrystals of ARTM with particle size of 161±1.5 nm and polydispersity index of 0.172±0.01 were produced in <1 hour using a wet milling technology, Dena® DM-100. The crystallinity of the processed ARTM was confirmed using differential scanning calorimetry and powder X-ray diffraction. The saturation solubility of the ARTM nanocrystals was substantially increased to 900 µg/mL compared to the raw ARTM in water (145.0±2.3 µg/mL) and stabilizer solution (300.0±2.0 µg/mL). The physical stability studies conducted for 90 days demonstrated that nanocrystals stored at 2°C–8°C and 25°C were very stable compared to the samples stored at 40°C. The nanocrystals were also shown to be stable when processed at acidic pH (2.0). The solubility and dissolution rate of ARTM nanocrystals were significantly increased (P<0.05) compared to those of its bulk powder form. The results of in vitro studies showed significant antimalarial effect (P<0.05) against Plasmodium falciparum and Plasmodium vivax. The IC50 (median lethal oral dose) value of ARTM nanocrystals was 28- and 54-fold lower than the IC50 value of unprocessed drug and 13- and 21-fold lower than the IC50 value of the marketed tablets, respectively. In addition, ARTM nanocrystals at the same dose (2 mg/kg) showed significantly (P<0.05) higher reduction in percent parasitemia (89%) against P. vivax compared to the unprocessed (27%), marketed tablets (45%), and microsuspension (60%). The acute toxicity study demonstrated that the LD50 value of ARTM nanocrystals is between 1,500 mg/kg and 2,000 mg/kg when given orally. This study demonstrated that the wet milling technology (Dena® DM-100) can produce smart nanocrystals of ARTM with enhanced antimalarial activities.