Ricardo Martins Duarte Byrro

Development and validation of a high‐performance liquid chromatography–electrospray ionization–MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma

A sensitive and fast high-performance liquid chromatography-electrospray ionization-MS/MS method for the simultaneous quantitation of levodopa and carbidopa in human plasma was developed and validated. A simple protein precipitation step with perchloric acid was used for the cleanup of plasma, and methyldopa was added as an internal standard. The analyses were carried out using an ACE C(18) column (50 × 4.6 mm i.d.; 5 µm particle size) and a mobile phase consisting of 0.2% formic acid and acetonitrile (90:10). The triple-quadrupole mass spectrometer equipped with an electrospray source in positive mode was set up in the selective reaction monitoring mode to detect the ion transitions m/z 198.1 → m/z 107.0, m/z 227.2 → m/z 181.0, and m/z 212.1 → m/z 139.2 for levodopa, carbidopa, and methyldopa, respectively. The method was validated and proved to be linear, accurate, and precise over the range 50-5000 ng/mL for levodopa and 3-600 ng/mL for carbidopa. The proposed method was successfully applied in a pharmacokinetic study with a levodopa/carbidopa tablet formulation in healthy volunteers.

Photodynamic therapy efficiently controls dermatophytosis caused by Trichophyton rubrum in a murine model

DEAR EDITOR, Dermatophytes are filamentous fungi that use keratin to colonize the host, causing superficial infections called dermatophytoses. Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum spp. and Epidermophyton spp. are important aetiological agents worldwide. A few drugs, including azoles (itraconazole), allylamines (terbinafine) and hydroxypiridones [ciclopirox olamine (CPX)], are available for the treatment of dermatophytoses, but the widespread use of antifungals has been linked to the emergence of resistant strains of T. rubrum. This problem may jeopardize treatment and thereby justifies the search for new therapeutic strategies. Antimicrobial photodynamic therapy (aPDT) combines a photosensitizer and a nonthermal light source to induce a reaction that results in cell death. In the presence of molecular oxygen, aPDT triggers the production of reactive oxygen (ROS) and reactive nitrogen species, which have short lifetimes and limited diffusion through the tissue, explaining the localized damage induced by aPDT. Previously, we have shown that T. rubrum is susceptible to photodynamic inhibition using toluidine blue (TBO) and light-emitting diode (LED) at 630 nm; however, there is a lack of studies regarding its efficacy in vivo. Therefore, the aim of this study was to evaluate the effects of aPDT, using TBO and LED, in controlling T. rubrum infection in a murine model of dermatophytosis. In this study, C57BL/6 mice were infected with 1 9 10 conidia per animal of T. rubrum American Type Culture Collection 28189 and treated with aPDT daily [using TBO 0 2% in a gel formulation (Sigma-Aldrich, St Louis, MO, U.S.A.) and 630-nm LED (Fisioled; MMoptics, S~ao Paulo, Brazil) at a dose of 42 J cm] or CPX [10 mg g 1 (dose of 0 65 mg 1 per mouse); Medley, S~ao Paulo, Brazil] for 48 h, over a period of 7 days (Comiss~ao de Etica no Uso de Animals/Universidade Federal de Minas Gerais ethics protocol approval no. 040/ 2011). Histopathology of the skin, fungal burden in potato agar, and myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activity were evaluated. The influence of aPDT on the viability of intraperitoneal macrophages, intracellular formation of hyphae and oxidative burst (ROS) was determined. For this purpose, TBO (10 lg mL ) and LED (48 J cm) were used. The groups of mice and macrophages used in this work are described in Table 1. Histopathological analyses showed that aPDT reduced the signs of dermatitis and recovered tissue architecture (Fig. 1c, g,i) compared with the infected and nontreated group (Fig. 1b,f,i). Interestingly, fungal cells were found mainly in hair follicles, corroborating the keratinophilic aspect of T. rubrum (Fig. 1f–h). Indeed, aPDT significantly reduced the fungal burden by 87% compared with the untreated group (P < 0 01) and by 64% compared with the CPX group (P < 0 05) (Fig. 1d,f–h,j). LED or TBO alone did not reduce the fungal burden (Fig. 1j). In addition, aPDT significantly

A rapid and sensitive HPLC–APCI-MS/MS method determination of fluticasone in human plasma: Application for a bioequivalency study in nasal spray formulations

A sensitive method for the determination of fluticasone in plasma was developed using high performance liquid chromatography with tandem mass spectrometric detection, whereas beclomethasone was used as internal standard. The analytes were extracted with a simple liquid-liquid extraction from the plasma samples and separated on an ACE C(18) 50 × 4.6 mm i.d.; 5 μm particle size column with a mobile phase consisting of acetonitrile - 0.01% formic acid (48:52, v/v) at a flow rate of 1 ml/min. Detection was achieved by an Applied Biosystems API 5000 mass spectrometer (LC-MS/MS) set at unit resolution in the multiple reaction monitoring mode. Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for fluticasone propionate was 85%, with a lower limit of quantification set at 2 pg/mL. The validated analytical method was applied to a bioequivalence study of fluticasone propionate administered by nasal spray formulations in human volunteers.

Simultaneous quantitation of levodopa and 3-O-methyldopa in human plasma by HPLC-ESI-MS/MS: application for a pharmacokinetic study with a levodopa/benserazide formulation.

A sensitive and simple method was developed for the quantitation of levodopa and its metabolite 3-O-methyldopa, in human plasma, after oral administration of tablet formulations containing levodopa (200 mg) and benserazide (50 mg). The analytes were extracted by a protein precipitation procedure, using carbidopa as an internal standard. A mobile phase consisting of 0.2% formic acid and acetonitrile (94:6, v/v) was used and chromatographic separation was achieved using ACE C(18) column (50 mm×4.6 mm i.d.; 5 μm particle size). Selected reaction monitoring was performed using the fragmentation transitions m/z 198→m/z 107, m/z 212→m/z 166 and m/z 227→m/z 181 for levodopa, 3-O-methyldopa and carbidopa, respectively. Calibration curves were constructed over the range 50.0-6000.0 ng/mL for levodopa and 25.0-4000.0 ng/mL for 3-O-methyldopa. The method shown to be specific, precise, accurate and provided recovery rates higher than 85% for all analytes. No matrix effect was detected in the samples. The validated method was applied in a pharmacokinetic study with a levodopa/benserazide tablet formulation in healthy volunteers.

Quality assurance of histamine analysis in fresh and canned fish.

Histamine determination is relevant for fish safety, quality and trade. Recently a study by the European Union (EU) compared the Codex and the EU mandated methods for the analysis of histamine and observed that they underestimated and overestimated the results, respectively. To solve this problem, a simple and efficient procedure for the extraction and quantification of histamine by ion-pair HPLC method with post-column derivatization and fluorimetric detection is proposed. It was optimized and validated for the analysis of histamine in fish. The method attended the performance criteria established by Commission Decision 2002/657/CE. The method was also submitted to proficiency testing; uncertainty was calculated; and the stability of solutions and standards was investigated. There was no matrix effect. The LOD, LOQ, CCα and CCβ were fit for the purpose. The method was successfully used in the analyses of freshwater fish and fresh and canned tuna.

Mucoadhesive chitosan films as a potential ocular delivery system for ofloxacin: preliminary in vitro studies

OBJECTIVE The objective of the study is to evaluate the physical properties, in vitro release profile, and antibacterial efficiency of chitosan films prepared with ofloxacin. PROCEDURE Mucoadhesive films were prepared by means of a casting and solvent evaporation technique performed in a 2 wt% acetic acid solution and distilled water. Physical properties were characterized by release and swelling studies, differential scanning calorimetry (DSC) analysis, and attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR) analysis. The in vitro evaluation of the films was performed by inhibiting Staphylococcus aureus and Pseudomonas aeruginosa through activity studies. RESULTS Circular ofloxacin-loaded chitosan-developed films with 0.3 mg of drug weighed 7 mg were 110 μm thick and 5 mm in diameter. The DSC curve of ofloxacin-loaded chitosan films suggests an amorphous dispersion of ofloxacin within these films. ATR-FTIR analysis showed that ofloxacin is indeed present in the matrix film. The drug was released in vitro over a 1-h period. No statistical difference could be observed between the ofloxacin-loaded chitosan films and sterile disk soaked for 1 min in 0.3% commercial ofloxacin ophthalmic solution for S. aureus and P. aeruginosa (P = 0.1686, P = 0.1172, respectively).The films presented a significantly larger mean bacterial inhibition zone of S. aureus than did the commercial ciprofloxacin control disk (P = 0.0002) and a significantly larger mean bacterial kill zone of P. aeruginosa than did the commercial enrofloxacin control disk (P < 0.0001). CONCLUSIONS Ofloxacin was successively incorporated onto chitosan films and was not inactivated during the process of manufacturing, thus preserving antibacterial proprieties.

Determination of Mycophenolic acid in the vitreous humor using the HPLC–ESI-MS/MS method: Application of intraocular pharmacokinetics study in rabbit eyes with ophthalmic implantable device

Mycophenolic acid (MPA) is an immunosuppressive agent widely used in the treatment of solid organ transplant rejection. The success of MPA in the treatment of inflammatory intraocular diseases has been reported in recent literature. The treatment of inflammatory eye diseases in the posterior chamber is a challenge due to the anatomy of the eye, which presents certain barriers to drug access. Thus, the bioavailability of drugs in the eye is quite low, and successful drug delivery may well represent a key limiting factor to attaining a successful therapeutic strategy. Ophthalmic controlled drug delivery offers the potential to enhance the efficacy of treatment for pathological conditions. Thus, a novel delivery system based on a biodegradable polymeric device, which can be implanted inside the eye and deliver MPA directly to the target, is being developed. Specific analytical methods to determine the use of effective drugs within the eye are needed to characterize this device. A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantitation of MPA in the vitreous humor of rabbits was developed and validated. The vitreous was collected from rabbits, extracted by a protein precipitation extraction procedure and then separated on a C18 column with a mobile phase comprised of 0.15% aqueous acetic acid and methanol (60:40, v/v). The calibration curve was constructed within the range of 3-10,000 ng/mL for MPA. The mean R.S.D. values for the intra-run and inter-run precision were 5.15% and 4.35%. The mean accuracy value was 100.16%. The validated method was successfully applied to determine the MPA concentration in the vitreous humor of rabbits treated with an ocular implantable device.

Quantitation of glucosamine sulfate in plasma by HPLC‐MS/MS after administration of powder for oral solution formulation

A rapid method for the quantification of glucosamine in human plasma using high-performance liquid chromatography coupled to tandem mass spectrometry was developed and validated. The sample preparation includes a simple deproteinization step, using D-[1-¹³C] glucosamine hydrochloride as an internal standard. Chromatographic separation was performed on an ACE Ciano column using isocratic elution with acetonitrile and aqueous 2 mM ammonium acetate containing 0.025% formic acid (80:20). Selected reaction monitoring was performed using the transitions m/z 180.1 → m/z 72.1 and m/z 181.0 → m/z 74.6 to quantify glucosamine and internal standard, respectively. The method was validated and proved to be linear, accurate and precise over the range 50-5000 ng/mL of glucosamine. Recovery rates higher than 90% were obtained for both glucosamine and internal standard. No matrix effect was detected in the samples. The validated method was successfully applied to a pharmacokinetic study after oral administration of a powder for oral solution formulation containing glucosamine sulfate.

Determination of triamcinolone in human plasma by a sensitive HPLC–ESI-MS/MS method: application for a pharmacokinetic study using nasal spray formulation

A liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantitation of triamcinolone in human plasma after nasal spray application was developed and validated. Betamethasone was used as internal standard (IS). The analytes were extracted by a liquid-liquid procedure and separated on a Zorbax Eclipse XDB C(18) column with a mobile phase composed of 2 mM aqueous ammonium acetate pH 3.2 and acetonitrile (55:45). Selected reaction monitoring was performed using the transitions m/z 435 → 415 and m/z 393 → 373 to quantify triamcinolone acetonide and betamethasone, respectively. Calibration curve was constructed over the range of 20-2000 pg/ml for triamcinolone acetonide. The lower limit of quantitation was 20 pg/ml. The mean RSD values were 4.6% and 5.7% for the intra-run and inter-run precision, respectively. The mean accuracy value was 98.5% and a recovery rate corresponding to 97.5% was achieved. No matrix effect was detected in the samples. The validated method was successfully applied to determine the plasma concentrations of triamcinolone acetonide in healthy volunteers, in a pharmacokinetic study with nasal spray formulation.

Clinical Response to Donepezil in Mild and Moderate Dementia: Relationship to Drug Plasma Concentration and CYP2D6 and APOE Genetic Polymorphisms

The clinical response to donepezil in patients with mild and moderate dementia was investigated in relation to the drug plasma concentration and APOE and CYP2D6 polymorphisms. In a prospective naturalistic observational study, 42 patients with Alzheimers disease (AD) and AD with cerebrovascular disease who took donepezil (10 mg) for 12 months were evaluated. Their DNA was genotyped, and the donepezil plasma concentrations were measured after 3, 6, and 12 months. Good responders scored ≥-1 on the Mini-Mental State Examination at 12 months in comparison to the baseline score. The study results indicated the good response pattern was influenced by the concentration of donepezil, but not by APOE and CYP2D6 polymorphisms.