Gold immunochromatographic strip assay for the detection of triamcinolone acetonide and budesonide in milk.

A monoclonal antibody against triamcinolone acetonide (TCA) and budesonide (BUD) was prepared using a hapten that was generated by introducing a carboxyl group into the structure of TCA. Based on the prepared monoclonal antibody, a gold nanoparticle-based lateral-flow immunoassay (GLFA) was developed with the ability to screen TCA and BUD in milk. The visible limits of detection of the GLFA for the analysis of TCA and BUD were 0.1 and 0.5 ng/mL with a cutoff value of 5 and 10 ng/mL, respectively, in milk. Average recoveries of TCA and BUD in milk were 92.0-102.2% and 96.0-98.8% with a good correlation between the results from the GLFA and LC-MS/MS analysis. These results demonstrated that the GLFA method for the rapid detection of TCA and BUD in milk samples is reliable and sensitive.

Sample preparation strategy for the detection of steroid-like compounds using MALDI mass spectrometry imaging: pulmonary distribution of budesonide as a case study.

Corticosteroids as budesonide can be effective in reducing topic inflammation processes in different organs. Therapeutic use of budesonide in respiratory diseases, like asthma, chronic obstructive pulmonary disease, and allergic rhinitis is well known. However, the pulmonary distribution of budesonide is not well understood, mainly due to the difficulties in tracing the molecule in lung samples without the addition of a label. In this paper, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging protocol that can be used to visualize the pulmonary distribution of budesonide administered to a surfactant-depleted adult rabbit. Considering that budesonide is not easily ionized by MALDI, we developed an on-tissue derivatization method with Girard's reagent P followed by ferulic acid deposition as MALDI matrix. Interestingly, this sample preparation protocol results as a very effective strategy to raise the sensitivity towards not only budesonide but also other corticosteroids, allowing us to track its distribution and quantify the drug inside lung samples.

Validation of a HPLC-UV method for the quantification of budesonide in skin layers.

A simple and sensitive HPLC method for the quantification of budesonide in skin layers was developed and validated. Budesonide was extracted from stratum corneum, epidermis and dermis by means of a mixture of acetonitrile:water (recovery > 90%). Budesonide quantification was performed with a RP-C18 column using methanol and water mixture (69:31, v/v) as mobile phase, pumped at 0.8 ml/min. The absorbance was monitored at 254 nm. The method resulted to be selective, linear in the range 0.05-5 or 10 µg/ml, precise and accurate. LLOQ resulted to be 0.05 µg/ml. The developed method appeared to be appropriate for the quantification of budesonide in skin layers at the end of in vitro permeation experiments since the recovery of the applied dose was 97 ± 1%, in line with requirement of the OECD guideline for the testing of the chemicals (Skin absorption: in vitro method).

An occupational exposure limit (OEL) approach to protect home healthcare workers exposed to common nebulized drugs.

Home healthcare is a growing area of employment. Assessment of occupational health risks to home health care workers (HHCWs) is important because in many cases the unique characteristics of the home environment do not facilitate the level of exposure control afforded to caregivers in hospitals and other fixed patient care sites. This assessment is focused on health risks to HHCWs from exposure to pharmaceutical drugs used to treat asthma and other respiratory diseases, which are commonly administered to patients in aerosolized form via nebulizers. We developed risk-based exposure limits for workers in the form of occupational exposure limits (OEL) values for exposure to nebulized forms of the three most common drugs administered by this method: albuterol, ipratropium, and budesonide. The derived OEL for albuterol was 2 µg/day, for ipratropium was 30 µg/day, and for budesonide was 11 µg/day. These OELs were derived based on human effect data and adjusted for pharmacokinetic variability and areas of uncertainty relevant to the underlying data (human and non-human) available for each drug. The resulting OEL values provide an input to the occupational risk assessment process to allow for comparisons to HHCW exposure that will guide risk management and exposure control decisions.

Simultaneous Determination of Formoterol Fumarate and Budesonide Epimers in Metered Dose Inhaler Using Ion-Pair Chromatography.

A simple, accurate and valid ion-pairing chromatographic method was developed for the simultaneous determination of formoterol fumarate (FF) and budesonide (BUD) epimers in metered dose inhaler. The separation was performed on C-18 column using mobile phase consisting of acetonitrile:0.05 M sodium acetate buffer (40:60% v/v) containing 0.03% sodium dodecyl sulfate adjusted to pH 3.1 using increasing volumes of either TEA or orthophosphoric acid isocratically eluted at 1.0 mL/min. Quantitation was achieved with UV detection at 214 nm. The retention times were 3.22, 6.41 and 6.91 min for formoterol fumarate, budesonide epimers B and A, respectively. The linearity range was 0.05-5.0 µg/mL for formoterol fumarate and 0.5-50.0 µg/mL for budesonide. The method was validated for, linearity; lower limit of quantification, lower limit of detection accuracy and precision. The proposed method is rapid (7 min), reproducible (RSD < 2.0%) and achieves satisfactory resolution between FF and BUD B (resolution factor = 12.07). The mean recoveries of the analytes in metered dose inhaler (99.97 and 99.83% for FF and BUD, respectively) were satisfactory.

Budesonide-loaded guar gum microspheres for colon delivery: preparation, characterization and in vitro/in vivo evaluation.

A novel budesonide (BUD) colon delivery release system was developed by using a natural polysaccharide, guar gum. The rigidity of the microspheres was induced by a chemical cross-linking method utilizing glutaraldehyde as the cross-linker. The mean particle size of the microspheres prepared was found to be 15.21 ± 1.32 µm. The drug loading and entrapment efficiency of the formulation were 17.78% ± 2.31% and 81.6% ± 5.42%, respectively. The microspheres were spherical in shape with a smooth surface, and the size was uniform. The in vitro release profiles indicated that the release of BUD from the microspheres exhibited a sustained release behavior. The model that fitted best for BUD released from the microspheres was the Higuchi kinetic model with a correlation coefficient r = 0.9993. A similar phenomenon was also observed in a pharmacokinetic study. The prolongation of the half-life (t1/2), enhanced residence time (mean residence time, MRT) and decreased total clearance (CL) indicated that BUD microspheres could prolong the acting time of BUD in vivo. In addition, BUD guar gum microspheres are thought to have the potential to maintain BUD concentration within target ranges for a long time, decreasing the side effects caused by concentration fluctuation, ensuring the efficiency of treatment and improving patient compliance by reducing dosing frequency. None of the severe signs, like the appearance of epithelial necrosis and the sloughing of epithelial cells, were detected.

Physicochemical compatibility of nebulizable drug admixtures containing budesonide and colistimethate or hypertonic saline.

Knowledge of the physicochemical compatibility of admixtures of nebulizable drugs is an important issue. In this article, the results of our recent study dealing with the compatibility of drug admixtures containing budesonide and colistin methanesulfonate (brand name Colistin CF) or budesonide and 5.85% sodium chloride solution are presented, as well as the up-to-date version of our compatibility table. Admixtures were prepared by mixing 2.0 mL Pulmicort either with 3.0 mL Colistin CF or 4.0 mL 5.85% sodium chloride solution. Test solutions were stored for 24 hours at room temperature under ambient light conditions. Physical compatibility was determined by measuring pH and osmolality. Concentrations of budesonide were measured by a high-performance liquid chromatography assay. The antibiotic activity of colistin methanesulfonate was determined in comparison to standard solutions using a microbiological assay. No loss in drug concentration of budesonide and no change in antibiotic activity of colistin methanesulfonate were detected over a test period of 24 hours. Osmolality remained unchanged in both types of admixtures. In admixtures of budesonide with colistin methanesulfonate, pH increased during the first 4 hours of storage, while in admixtures of budesonide and hypertonic saline pH remained unchanged. No visible changes could be detected. Due to these results admixtures of budesonide and colistin methanesulfonate or 5.85% sodium chloride solution are designated to be compatible, but it is recommended that mixing should take place immediately before administration. Further investigations are needed to determine whether or not drug delivery is affected by mixing the drugs and to ensure simultaneous nebulization is recommendable.

The determination of budesonide and fluticasone in human sputum samples collected from COPD patients using LC-MS/MS.

A bioanalytical method for the quantitative determination of budesonide and fluticasone in human sputum was developed. Sputolysin(®) Reagent was added to the sputum samples. After incubation (37°C; 60-70 min under shaking) and automated solid phase extraction the extracts were analysed using LC-MS/MS. Budesonide and fluticasone showed good linearity (r>0.99) over the range 0.1-100 nM in the first and second validation batch, and over the range 0.25-10,000 nM in the third and fourth validation batch. The lower limit of quantification (LLOQ) achieved was 5 nM for budesonide and fluticasone in 100 µL human sputum. Intra-run and inter-run RSD for four quality control levels (5-100 nM) were within 6.9% (budesonide) and 8.0% (fluticasone). The accuracy ranged from -11.4% to -1.6% (budesonide), and from -11.8% to 0.4% (fluticasone). The validated method was applied to clinical sputum samples from COPD patients.

Inhalation performance of pollen-shape carrier in dry powder formulation: effect of size and surface morphology.

In a previous study, pollen-shape drug carriers are compared with traditional carriers at different drug mixing ratios and flow rates. It is found that pollen-shape drug carriers can deliver large amount of drug particles and reduce drug losses especially at low flow rates and high drug mixing ratios. In this study, the effect of size and surface morphology of pollen-shape carriers on drug delivery performance is assessed. Pollen-shape carrier particles having various sizes and surface asperities are synthesized. Budesonide (Bd) is used as the model drug. The drug delivery performances of the pollen-shape carrier particles are investigated using an Andersen Cascade Impactor (ACI) equipped with a Rotahaler at gas flow rates of 30 and 60 L/min. Three drug mixing ratios are considered. While an increase in the carrier particle size has a mild improvement on the ED, it significantly improves the FPF. A sparse surface asperity has negligible effect on the ED at low flow rates but it improves the FPF compared to a dense surface asperity under all experimental conditions.

Development and validation of a stability-indicating high-performance liquid chromatography method for the simultaneous determination of albuterol, budesonide, and ipratropium bromide in compounded nebulizer solutions.

In recent years, there has been a large increase in the use of pharmaceutical compounding to prepare medications that are not commercially available. The treatment of asthma typically includes the use of albuterol (ALB), ipratropium bromide (IPB), and/or budesonide (BUD) nebulizer solutions. There is currently no commercially available nebulizer solution containing all three of these compounds, and patients must rely on often-unregulated compounding. There is a distinct need for methodologies that can be used to analyze compounded formulations to ensure patient safety. We report an HPLC-UV method to separate and quantitate ALB, IPB, and BUD in nebulizer solutions. The method used a gradient elution to achieve separation via an RP C18 column. The method was validated, showed good selectivity, and was linear over several orders of magnitude. The method was applied to the analysis of nebulizer solutions and determination of their storage stability. Significant ALB-dependent degradation occurred within 5 h in solutions formulated with the free base of ALB, while those containing the sulfate salt of ALB produced no degradation. Alkali solutions can cause base-catalyzed hydrolysis of IPB and degradation of BUD. Compounded formulations containing ALB need to include an acid to control pH and prevent degradation.

A new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to evaluate pulmonary drug absorption for metered dose dry powder formulations.

Absorption studies with aerosol formulation delivered by metered dose inhalers across cell- and tissue-based in vitro models of the pulmonary epithelia are not trivial due to the complexity of the processes involved: (i) aerosol generation and deposition, (ii) drug release from the carrier, and (iii) absorption across the epithelial air-blood barrier. In contrast to the intestinal mucosa, pulmonary epithelia are only covered by a thin film of lining fluid. Submersed cell culture systems would not allow to studying the deposition of aerosol particles and their effects on this delicate epithelial tissue. We developed a new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to mimic the inhalation of a single metered aerosol dose and its subsequent deposition on filter-grown pulmonary epithelial cell monolayers exposed to an air-liquid interface. The reproducibility of deposition of these dry powder aerosols and subsequent drug transport across Calu-3 monolayers with commercially available dry powder inhalers containing salbutamol sulphate or budesonide could be demonstrated. In the context of developing new dry powder aerosol formulations, PADDOCC appears as a useful tool, allowing reducing animal testing and faster translation into clinical trials.

Aerosol delivery of nebulised budesonide in young children with asthma.

BACKGROUND: Lung deposition of inhaled steroids, likely to be of benefit in the anti-inflammatory treatment of asthma in young children, is low. This is explained by age specific anatomical and physiological characteristics as well as poor cooperation with aerosol therapy. However, total lung deposition and the ratio of lung deposition to oropharyngeal deposition are key determinants of clinical efficacy and of systemic side effects of aerosolized drugs. OBJECTIVES: The aim of this study was to determine lung deposition and ratio of lung deposition to oropharyngeal deposition using a modified vibrating membrane nebuliser to deliver budesonide with a small particle size, taking into account the needs of young children. PATIENTS AND METHODS: Ten asthmatic children (5 males), mean age 20.3 months (range 6-41 months) inhaled radiolabelled budesonide (MMD 2.6microm) through a modified vibrating membrane nebuliser (modified PARI e-Flow). Lung deposition expressed as a percentage of the emitted dose was measured using scintigraphy and the ratio of lung deposition to oropharyngeal deposition was calculated. RESULTS: Mean lung deposition (SD) expressed as percentage of emitted dose and mean lung to oropharyngeal deposition ratio (SD) in quietly breathing children (n=5) and in children crying during inhalation were 48.6% (10.5) versus 20.0% (10.9), and 1.0 (0.3) versus 0.3 (0.2), respectively. CONCLUSIONS: We have shown that by using an improved age-adjusted complementary combination of delivery device and drug formulation to deliver small particles, lung deposition and ratio of lung deposition to oropharyngeal deposition in young asthmatic children is highly improved. But the main factor limiting aerosol delivery in this age group remains cooperation.

Pitfalls and success of experimental design in the development of a mixed MEKC method for the analysis of budesonide and its impurities.

A mixed MEKC method for the analysis of budesonide and its related substances is presented. The micelles were formed from sodium cholate (CHOL) and 3-(N,N-dimethylmyristylammonio)propanesulfonate (MAPS). A multivariate optimisation was carried out with the aim of obtaining a baseline separation of all compounds. The influence of voltage, borate concentration, cholate concentration, MAPS concentration and pH was evaluated on the responses, corresponding to critical resolution values. Problems with the investigated experimental design were encountered due to the complexity of the separation process. As a consequence, a first design was not sufficient to reach the optimal conditions, but was needed in order to obtain the necessary information to successfully plan a second in-depth study by means of response surface methodology. The optimal conditions were as follows: capillary total and effective lengths of 48.5 and 40.0 cm, respectively, with 50 microm id; 70 mM borate buffer (pH 8.8) containing 65 mM CHOL and 10 mM MAPS; temperature 20 degrees C and voltage 16 kV. Separation of all the compounds, including R- and S-epimers of budesonide, was obtained in a reasonable time. Validation of the method was performed for both drug substances and drug product.

Estimation and characterisation of budesonide tablets impurities.

Budesonide is the 16alpha,17alpha-acetal of 16alpha-hydroxyprednisolone with n-butyraldehyde, endowed with anti-inflammatory activity. In a sample of budesonide tablets, kept for 3 years at 25 degrees C and 60% RH unknown impurities, not reported in European Pharmacopoiea, were present. Their identification was achieved by means of chemical and spectroscopic methods.

Development, evaluation and data acquired with a tape-stripping technique for measuring dermal exposure to budesonide at a pharmaceutical manufacturing site.

OBJECTIVES: Although corticosteroids have been used for over 50 years as anti-inflammatory and anti-proliferative agents, few studies have examined their exposure levels and health effects on workers employed in the corticosteroid manufacturing industry. The aims of the study reported here were to develop a tape-stripping technique for monitoring budesonide (a corticosteroid used in inhalators for treating respiratory diseases) and to apply the method in a pilot study to estimate the potential dermal exposure to budesonide among workers at a pharmaceutical formulation site. METHODS: The tape-stripping method was evaluated by applying 0.5 and 2.07 microg of budesonide dissolved in ethanol on tape strips. The same amounts were also applied on a cleaned glass plate and human skin of volunteers, which were then stripped by series of tapes immediately, and 30 min later, the amounts collected by the tapes were measured. Finally, the technique was used to study the exposure of budesonide among eight employees at a pharmaceutical industry site. Three exposure sites were tested: the tip of the forefinger, palm of the hand and ventral part of the lower arm. Five consecutive tape strips per sampling site were used in both the recovery studies and the field study. RESULTS: The mean overall recoveries from spiked tapes and the glass plate were 96 and 81%, respectively, while for human skin the corresponding figure was 38%, (for applications of 2.07 microg; no detectable amounts were recovered from human skin after 0.5 microg applications). The recovered amount was found on two consecutive tapes after 0 min, but only on the first tape strip after 30 min. The inter-individual variability was 4-fold. In the field, quantifiable amounts were found for four of eight employees and a concentration gradient was detected along the two or three consecutive tape strips. The tip of the forefinger and the palm of the hand were the most highly exposed sites to budesonide. CONCLUSIONS: A tape-stripping method can be used to determine potential dermal exposure to budesonide. The results also indicate that budesonide is taken up by the skin of operators who are exposed to the substance at their workplace.

Development and validation of a high-performance liquid chromatographic method for the analysis of budesonide.

A simple, rapid, and stability indicating reversed-phase high-performance liquid chromatography (HPLC) method of analysis for budesonide, a novel glucocorticoid prescribed for inflammatory bowel disease, was successfully developed. Budesonide is an epimeric mixture and both the epimers have similar anti-inflammatory activity. All the analytical methods reported in the literature are long and are based on separation of the epimers, thus our objective was to obtain a single sharp peak of the drug and to separate the drug peak from all the other degradation products. The method, was used to quantify budesonide in the developed formulation, employed a Kromasil C8, (150 mm x 4.6 mm) column with an isocratic mobile phase of acetonitrile-phosphate buffer (pH 3.2-0.025 M) (55:45 v/v), at a flow rate of 1.1 mL/min. Budesonide was detected by an ultraviolet detector at 244 nm. The method was validated for linearity, precision, repeatability, sensitivity, and selectivity. Selectivity was validated by subjecting stock solution of budesonide to acidic, basic, oxidative, and thermal degradation. The retention time of budesonide was about 4 min with symmetrical peaks. The method was linear over a concentration range 1-50 microg/mL (R2=0.9995). The limit of detection of budesonide was 0.1 microg/mL and the limit of quantitation was 0.25 microg/mL. The peaks of the degradation products did not interfere with the peak of budesonide. The developed method was used to quantify budesonide in budesonide-loaded micro-particles. Excipients present in the micro-particles did not interfere with the analysis and the recovery of budesonide from micro-particles was quantitative.

High performance liquid chromatography assay method for simultaneous quantitation of formoterol and budesonide in Symbicort Turbuhaler.

A sensitive and rapid high performance liquid chromatography method has been developed and used for the simultaneous determination of formoterol and budesonide in Symbicort Turbuhaler when assessing the aerodynamic characteristics of the emitted dose using Pharmacopoeial methods. This capability results in both time and cost saving. The mobile phase composition was acetonitrile-5 mM sodium dihydrogen orthophosphate, pH 3 (60: 40% v/v), and was passed at 1.5 ml min(-1) through a C18 column with a UV detection (wavelength 214 nm). The method was shown to give good analytical performance in terms of linearity, precision (using phenylpropanolamine as an internal standard), sensitivity and solution stability. The intra-day precision for both formoterol and budesonide were 0.75% and 1.11%, respectively (n = 10). The limit of quantitation for formoterol was 10 microgL(-1) and for budesonide was 120 microgL(-1), and the limit of detection were 3 and 30 microgL(-1), for both formoterol and budesonide, respectively. The method has been applied to determine the content of the emitted dose and the fine particle dose of Symbicort Turbuhaler.

Chromatographic and mass spectral characterization of budesonide and a series of structurally related corticosteroids using LC-MS.

The LC-MS characteristics of budesonide and a series of structurally related corticosteroids were reviewed to commence the construction of a library of chromatographic and mass spectral information to aid identification of budesonide degradation products during formulation stabilization investigations. The LC-ESI(+)-MS technique employing a Hypersil C18 column with a mobile phase of ethanol-acetonitrile-formic acid (pH 3.8; 0.14 mM) (2:30:68, v/v/v) was then used to characterize 23 corticosteroids. Based on their structures, the corticosteroids were classified into three groups: (I) 4-pregnene-3-one steroids; (II) 1,4-pregnadien-3-one steroids with no fluorine substituents; and (III) 1,4-pregnadiene-3-one steroids with fluorine substituents. Chromatographic (retention time and UV absorbance) and mass spectral properties were correlated with the known chemical structures of these corticosteroids. Base peak and mass spectral fragmentation patterns were related to steroid structural characteristics.

Surface energy and interparticle forces correlations in model pMDI formulations.

PURPOSE: To compare experimental measurements of particle cohesion and adhesion forces in a model propellant with theoretical measurements of the interfacial free energy of particulate interactions; with the aim of characterizing suspension stability of pressurized metered dose inhalers (pMDIs). METHODS: Interparticulate forces of salbutamol sulfate, budesonide, and formoterol fumarate dihydrate were investigated by in situ atomic force microscopy (AFM) in a model propellant 2H,3H perfluoropentane. The surface thermodynamic properties were determined by contact angle (CA) and inverse gas chromatography (IGC). Experimental data were compared with theoretical work of adhesion/cohesion using a surface component approach (SCA), taking into account both dispersive and polar contributions of the surface free energy. RESULTS: Results indicated that the measured forces of interaction between particles in model propellant could not be accounted for by theoretical treatment of the dispersive surface free energies via CA and IGC. A correlation between theoretical work of adhesion/cohesion and AFM measurements was observed upon the introduction of the polar interfacial interactions within the SCA model. CONCLUSIONS: It is suggested that the polar contributions of the surface free energy measurements of particles may play a crucial role in particle interaction within propellant-based systems. Together with the application of a SCA model, this approach may be capable of predicting suspension stability of pMDI formulations.

Lower oropharyngeal deposition of inhaled ciclesonide via hydrofluoroalkane metered-dose inhaler compared with budesonide via chlorofluorocarbon metered-dose inhaler in healthy subjects.

OBJECTIVE: Inhaled corticosteroids may cause oropharyngeal side effects if deposited in the oropharynx in active form. Ciclesonide, an inhaled corticosteroid with low glucocorticoid receptor affinity, is activated primarily in the lung by esterases to an active metabolite, desisobutyryl-ciclesonide (des-CIC), with high glucocorticoid receptor affinity. We studied oropharyngeal deposition of ciclesonide, des-CIC, and budesonide. METHODS: In an open-label, randomized, two-treatment (administered in sequence), five-period study, 18 healthy subjects received 800 microg (ex-valve) inhaled ciclesonide via a hydrofluoroalkane-pressurized, metered-dose inhaler followed by 800 microg budesonide (Pulmicort) by a chlorofluorocarbon-pressurized, metered-dose inhaler (four puffs of 200 microg each, ex-valve) or vice versa. Oropharyngeal cavity rinsing was performed immediately, or 15, 30, 45, or 60 min after inhalation (one rinsing per study period), and the solutions were analyzed using liquid chromatography with tandem mass spectrometric detection. RESULTS: Ciclesonide and budesonide were detected in most oropharyngeal wash samples. Maximal concentration of each inhaled corticosteroid was reached immediately post-inhalation; maximal concentrations of ciclesonide and des-CIC were 30% and 0.67%, respectively, of budesonide. Oropharyngeal deposition of ciclesonide and budesonide decreased rapidly within 15 min post-inhalation, and less rapidly thereafter. Less than 10% of the residual ciclesonide in the oropharynx was converted to des-CIC. The molar dose-adjusted amount of des-CIC was 4% of budesonide (P < 0.0001). There were no significant adverse events. CONCLUSION: Oropharyngeal deposition of des-CIC was more than one order of magnitude lower than that of budesonide when administered by the respective metered-dose inhalers. This may explain the low frequency of oropharyngeal side effects of ciclesonide in clinical studies.