Rapid analysis of aerosol drugs using nano extractive electrospray ionization tandem mass spectrometry.

Aerosol drugs dominate a significant share of pharmaceutical preparations on the market. A novel sensitive method utilizing nano extractive electrospray ionization mass spectrometry (nanoEESI-MS) has been developed for the rapid analysis of aerosol drug samples with quantitative information. Without any sample pretreatment, aerosol drugs were manually sprayed into the primary ion plume created by a nano electrospray emitter for direct ionization under ambient conditions. The analyte ions of interest were guided into an ion trap mass spectrometer for tandem mass analysis. The active ingredients of various aerosol drugs, such as econazole nitrate, beclomethasone dipropionate, binary mixture of methyl salicylate and diphenhydramine, terbutaline, and salbutamol, were rapidly detected using nanoEESI-MS. A single sample analysis could be completed within 1.2 s. Tandem mass spectrometry was used to confirm the identification of important compounds in each aerosol drug sample. Reasonable relative standard deviation (RSD = 6.39%, n = 13) and acceptable sensitivity (10 ppt, 100 muL) were found for the salbutamol aerosol sample, which suggests that nanoEESI-MS has the quantitative capacity for analyzing complex pharmaceutical samples. This method was further extended to study the thermal decomposition process of salbutamol, showing that the degradation kinetics of salbutamol can be conveniently tracked. Our data demonstrate that nanoEESI tandem mass spectrometry is a fast and sensitive technique for the analysis of aerosol drug preparations, showing promising applications in pharmacology studies and in situ analysis of aerosol drugs on the market.

Identification and characterization of unknown degradation impurities in beclomethasone dipropionate cream formulation using HPLC, ESI-MS and NMR.

The present study focuses on identifying the degradation profile and pathways of unknown impurities from beclomethasone dipropionate (BDP) topical cream formulation reported under accelerated stability conditions. Six degradation impurities were observed during the accelerated stability testing of BDP topical cream formulation, and these thermally labile degradation impurities were primarily identified using a simple, effective and mass compatible isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection method. The degradation impurities found in this sample were of very low concentration levels, thus the concentration of these impurities in the sample was enriched by mimicking the thermal degradation conditions to structurally elucidate the unknown impurities. These BDP thermal degradation impurities were isolated using preparative liquid chromatography and followed by pre-concentration using rota-vapour. Further, the collected thermal degradation impurities were characterized using ESI-MS, and the major impurity was identified using 1H and C13 NMR spectroscopy, and DEPT technique. Plausible degradation pathway and mechanism of each impurity from BDP has been proposed based on the obtained mass and NMR spectral data. Thus, the present method is simple and suitable to be applied towards BDP assay in various formulations, and also to investigate the thermal stability and degradation kinetics of the final drug product.

Quantitative determination of drug encapsulation in poly(lactic acid) nanoparticles by capillary electrophoresis.

Capillary electrophoretic (CE) methods were used for the quantitative determination of model drugs [salbutamol sulphate (SS), sodium cromoglycate (SCG) and beclomethasone dipropionate (BDP)] in poly(D,L-lactic acid) (PLA) nanoparticles, which were prepared by the nanoprecipitation method. Zeta potential and size distribution of the nanoparticles were determined by electrophoretic mobility determinations and photon correlation spectroscopy, respectively. Interactions between the drugs, the PLA nanoparticles and the fused-silica capillary were investigated by electrokinetic capillary chromatography (EKC). A quantitative CE method was developed for salbutamol sulphate and sodium cromoglycate, and the linearity and repeatability of migration times, peak areas and peak heights were determined. Microemulsion electrokinetic chromatography was used for the quantitative determination of beclomethasone dipropionate. According to this study, the applied electromigration techniques were suitable for the interaction, drug entrapment and dissolution studies of pharmaceutical nanoparticles. The results suggest that even quantitation of the drug located inside the nanoparticles was possible. Encapsulation of the more hydrophilic model drugs (SS, SCG) in the PLA nanoparticles was less efficient than in the case of BDP.

A comparison of two methods to determine the solubility of compounds in aerosol propellants.

A new on-line reverse phase HPLC method for determining the solubility of compounds in propellant based metered dose inhaler (MDI) formulations was compared with a conventional method. The new method employs a direct injection from a MDI vial into the needle injector port of a manual injector. To evaluate the two methods, beclomethasone dipropionate (BDP), 5,5-diphenyl hydantoin and 3,3'-diindolylmethane, were used as model compounds in propellant HFA-134a. Comparison was performed by analyzing known and unknown concentrations of BDP in various combinations of HFA-134a and ethanol. In addition, the solubility of 5,5-diphenyl hydantoin and 3,3'-diindolylmethane were determined in HFA-134a using both the new and the conventional methods. The two methods were found to be in good agreement with each other, with the new direct injection technique offering enhanced precision and accuracy along with considerable reduction in analysis time.

High-pressure aerosol suspensions--a novel laser diffraction particle sizing system for hydrofluoroalkane pressurised metered dose inhalers.

In this study, a novel laser diffraction particle size analysis dispersion system, capable of sizing particles in situ within suspension hydrofluoroalkane (HFA) pressurised metered dose inhalers (pMDIs), was developed and tested. The technique was compared to four indirect particle sizing methods commonly used to determine the size of particles suspended in HFA pMDIs. The median volume diameter obtained using laser diffraction of both the salbutamol sulphate and fluticasone propionate suspended either in 2H, 3H-decafluoropentane or perfluoropentane (employed as surrogate propellants) was over one-order of magnitude larger than the particle sizes of the drugs suspended in HFA 134a. In contrast, the "in-flight" particle size using the Sympatec inhaler 2000 laser diffraction equipment undersized the particles, predicting higher delivery efficacy compared to the other sizing methods. However, the size of particles suspended in HFAs derived using the novel pressurised dispersion system, showed a linear correlation with the impaction results, r2=0.8894 (n=10). The novel pressure cell sizing technique proved to be simple to use, has the ability to be automated and was accurate, suggesting it could be an essential tool in the development of new suspension-based pMDI formulations.

Proliposome powders prepared using a slurry method for the generation of beclometasone dipropionate liposomes.

A novel "slurry method" was described for the preparation of proliposome powders using soya phosphatidylcholine (SPC) with cholesterol (1:1) and for incorporation of beclometasone dipropionate (BDP) at 2mole% of the total lipid phase. Proliposomes made with a range of lipid to sucrose carrier ratios were studied in terms of surface morphology using scanning electron microscopy (SEM) and thermal properties using differential scanning calorimetry (DSC). Following hydration of proliposomes, the resultant vesicles were compared to liposomes made using the traditional proliposome method, in terms of vesicle size and drug entrapment efficiency. SEM showed that sucrose was uniformly coated with lipid regardless of lipid to carrier ratio. Liposomes generated using the slurry proliposome method tended to have smaller median size than those generated with the conventional proliposome method, being in the range of 4.72-5.20µm and 5.89-7.72µm respectively. Following centrifugation of liposomes using deuterium oxide (D2O) as dispersion medium, vesicles entrapping BDP were separated as a floating creamy layer, whilst the free drug was sedimented as crystals. Drug entrapment was dependent on formulation composition and preparation method. When 1:15 w/w lipid to carrier was used, liposomes generated using the slurry method had an entrapment efficiency of 47.05% compared to 18.67% for those generated using the conventional proliposome method. By contrast, liposomes made by the thin-film hydration method had an entrapment efficiency of 25.66%. DSC studies using 50mole% BDP demonstrated that the drug was amorphous in the proliposome formulation and tended to crystallize on hydration, resulting in low drug entrapment. In conclusion, a novel approach to the preparation of proliposomes using a slurry method has been introduced, offering higher entrapment for BDP than liposomes made using the conventional proliposome method and those prepared by thin-film hydration technique.

Operating characteristics of 18 different continuous-flow jet nebulizers with beclomethasone dipropionate liposome aerosol.

A study of 18 different commercially available continuous-flow, jet nebulizers was performed with a standard liposomal formulation of beclomethasone dipropionate (Bec-DP) prepared with dilauroyl phosphatidylcholine (Bec-DLPC). The analysis compared the total Bec-DP output from aqueous suspensions of Bec-DLPC containing an initial starting reservoir concentration of 0.5 mg/ml. Aerosols from each nebulizer tested were characterized by the mass median aerodynamic diameter, geometric standard deviation, drug output, and the predicted percentage regional deposition of inhaled Bec-DLPC liposomes within the human respiratory tract. These data can provide a basis for the selection of commercially available jet nebulizers for use with glucocorticoid liposome aerosols for treatment of asthma and other inflammatory lung diseases.

Sensitive liquid chromatographic/tandem mass spectrometric method for the determination of beclomethasone dipropionate and its metabolites in equine plasma and urine.

Beclomethasone dipropionate (BDP) is a potent pro-drug to beclomethasone (BOH) and is used in the treatment of chronic and acute respiratory disorders in the horse. The therapeutic dose of BDP (325 microg per horse) by inhalation results in very low plasma and urinary concentrations of BDP and its metabolites that pose a challenge to detection and confirmation by equine forensic laboratories. To solve this problem, a method involving the use of a liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the detection, confirmation and quantification of the analytes in equine samples. Ammonium formate or acetate buffer added to LC mobile phase favored the formation of [M + H](+) ions from BDP and its metabolites, whereas formic acid led to the formation of sodium and potassium adduct ions ([M + Na](+), [M + K](+)) together with [M + H](+) ions. Acetonitrile, on the other hand, favored the formation of abundant solvent adduct ions [M + H + CH(3)CN](+) with the analytes under electrospray ionization (ESI) and atmospheric pressure chemical ionization conditions. In contrast, methanol formed much less solvent adduct ions than acetonitrile. The solvent adduct ions were thermally stable and could not be completely desolvated under the experimental conditions, but they were very fragile to collision-induced dissociation (CID). Interestingly, these solvent adduct ions were observed on a triple-quadrupole mass spectrometry but not on an ion trap instrument where helium used as a damping gas in the ion trap might cause the solvent adduct ions desolvated by collision. By CID studies on the [M + H](+) ions of BDP and its metabolites, their fragmentation paths were proposed. In equine plasma at ambient temperature over 2 h, BDP and B21P were hydrolyzed in part to B17P and BOH, respectively, but B17P was not hydrolyzed. Sodium fluoride added to equine plasma inhibited the hydrolysis of BDP and B21P. The matrix effect in ESI was evaluated in equine plasma and urine samples. The method involved the extraction of BDP and its metabolites from equine plasma and urine samples by methyl tert-butyl ether, resolution on a C(8) column with a mobile phase gradient consisting of methanol and ammonium formate (2 mmol l(-1), pH 3.4) and multiple reaction monitoring for the analytes on a triple-quadrupole mass spectrometer. The detection limit was 13 pg ml(-1) for BDP and B17P, 25 pg ml(-1) for BOH and 50 pg ml(-1) for B21P in plasma and 25 pg ml(-1) for BOH in urine. The method was successfully applied to the analysis of equine plasma and urine samples for the analytes following administration of BDP to horses by inhalation. B17P, the major and active metabolite of BDP, was detected and quantified in equine plasma up to 4 h post-administration by inhalation of a very low therapeutic dose (325 microg per horse) of BDP.

Use of detergents and high contents of organic solvents for simultaneous quantitation of ionic and nonionic drugs by electrokinetic chromatography.

Buffers containing high percentages of organic solvents, typically 50% of acetonitrile and/or methanol, together with sodium dodecyl sulfate (SDS) are employed for the separation and quantitation by electrokinetic chromatography (EKC) of analytes found in a nasal spray. Solutes consist of benzalkonium chloride, a family of highly positive compounds, and 2-phenylethanol and beclomethasone dipropionate, which are electrically neutral and poorly soluble in aqueous buffers. It is observed that the effect of both concentration of SDS and temperature on the separation depends on the organic solvent used and the solute nature. It is also observed that SDS-solute interaction for neutral and cationic compounds are weaker in the presence of high contents of acetonitrile than in methanol. Concentration of SDS, temperature, and organic solvent nature and content, allow one to modify the selectivity of the separation when neutral and ionic species have to be simultaneously determined. The optimization of EKC conditions enables the analysis of compounds in less than 5 min. A one-step sample treatment consisting of centrifugation of the nasal spray solved in acetonitrile, together with the referenced optimum separation conditions enable the reproducible quantitation of the analytes. Relative standard deviation values of inter-day migration times lower than 2.45% are obtained (R.S.D.n = 12), while R.S.D.n = 12 values for inter-day peak areas were lower than 6.32%.

Quantitation of active ingredients and excipients in nasal sprays by high-performance liquid chromatography, capillary electrophoresis and UV spectroscopy.

A study on the use of different analytical methodologies to determine active ingredients and excipients found in commercial nasal sprays is presented. Two of the developed methodologies consisted of separation techniques, i.e. high-performance liquid chromatography and capillary electrophoresis, and the third one involved a UV-spectroscopic multicomponent procedure. The samples studied are characterized by a high viscosity and the existence of a large number of particles in suspension; therefore, special emphasis is paid on the sample preparation required by each methodology. Advantages and drawbacks of each analytical technique are also discussed in terms of speed of analysis, sensitivity and reproducibility. From this work it is observed that although the UV method needs the most laborious sample preparation, the total time required per analysis is the shortest one. The best reproducibility in terms of analysis time and quantitation of the analyzed compounds is obtained using HPLC. CE allows the determination of more components in the same sample.

Aerosolisation of beclomethasone dipropionate using spray dried lactose/polyethylene glycol carriers.

The aim of this study was to characterize the physical properties of spray dried lactose in the presence of different polyethylene glycols (PEG 400, PEG 3000 and PEG 6000) and to evaluate their performance as carriers for dry powder inhaler (DPI) formulations. The efficiency of spray dried lactose/PEG carriers in aerosolisation of beclomethasone dipropionate (BD), a model hydrophobic drug, was compared to Pharmatose 325 M (L325), spray dried lactose alone (SDL), and also a sieved (< 38 microm) fraction of alpha-lactose monohydrate (SL). In vitro deposition analysis was performed using a twin stage liquid impinger at a flow rate of 60 l/min through a Spinhaler. The deposition profiles of the drug from binary formulations composed of BD and spray dried lactose/PEG carriers were also compared to ternary formulations containing large and fine lactose carriers. Differential scanning calorimetry and X-ray diffraction data showed the presence of alpha-anhydrous lactose in spray dried lactose/PEG crystalline powders. Spray drying of lactose in the presence of PEG 400 resulted in the production of a powder (SDL-PEG400) with lower alpha-lactose monohydrate content, and also smaller particle size distribution than those obtained in the presence of PEG 3000 (SDL-PEG3000) or PEG 6000 (SDL-PEG6000). All formulations showed different deposition profiles, except those containing SDL-PEG3000 or SDL-PEG6000 which exhibited similar data. The fine particle fraction of aerosolised BD varied from 6.26 +/- 1.07 (for L325) to 25.87 +/- 5.33 (for SDL-PEG3000). All deposition profiles of BD aerosolised from SDL-PEG3000 were significantly higher (P < 0.01) than those produced by binary and ternary formulations containing L325, a coarse lactose commercially available for DPI formulations. The differences observed in deposition data for various carriers were interpreted according to their physical properties. It was concluded that particle size distribution, morphology and specific surface texture of SDL-PEG3000 and SDL-PEG6000 were important factors influencing their efficiency as small carriers for DPI formulations.

Equivalence testing and equivalence limits of metered-dose inhalers and dry powder inhalers measured by in vitro impaction.

In this study, criteria for the acceptability of comparative in vitro equivalence testing are proposed. Furthermore, the following equivalence limits for in vitro impaction methods are postulated: the 90% confidence interval (CI) of the in vitro deposition ratio of the test product and the reference product should lie within 0.80-1.20. The aim of this study was to challenge these limits by applying them to in vitro impaction results of several groups of pressurized metered-dose inhalers and dry powder inhalers containing salbutamol and beclomethasone dipropionate. The deposition results were obtained with the Twin Impinger. All products had a marketing authorization in The Netherlands and were considered therapeutically equivalent within each group. The postulated equivalence limits/group were challenged by fictitiously assigning a preparation as a test product or reference product and calculating the 90% CI of the deposition ratio of the test and reference products. All possible combinations of products within a group were tested. The products were considered equivalent if the 90% CI of the quotient lay within 0.80-1.20. In most cases, the quotient of the test product and reference product remains within 0.80-1.20, but due to a high variability in the deposition results of several products, the 90% CI of the quotient sometimes falls outside the proposed equivalence limits. It is concluded that the equivalence limits postulated are rather conservative, with respect to accepting equivalence. The limits can therefore serve as a prudent predictor of equivalence within the acceptability criteria proposed, but have to be further validated.

The measurement of beclomethasone dipropionate entrapment in liposomes: a comparison of a microscope and an HPLC method.

The purpose of this study was to examine the methodologies that may be used to estimate the maximum incorporation (<5 mole% drug) of beclomethasone dipropionate (BDP) in dipalmitoylphosphatidylcholine (DPPC) multilamellar liposomes. Two approaches are described. First, differential interference contrast (DIC) microscopy and cross-polarisation microscopy have been used to measure the concentration at which BDP crystals become apparent in BDP-containing liposome preparations, thereby allowing a semi-quantitative but simple estimation of entrapment. An alternative method is described whereby the unentrapped solid drug is separated from the liposomes via suspension in D2O, followed by centrifugation and HPLC analysis. The method resulted in an estimate of 1.5-2 mole% BDP, while the HPLC method yielded a value of 2.52 mole% BDP.

First-derivative spectrophotometric determination of salbutamol in pharmaceutical preparations.

The development of a first-order derivative spectrophotometric assay of salbutamol as a single-component and in combination with beclomethasone dipropionate in pharmaceutical formulations is described. The method eliminates the interference of tablet excipients and allows the determination of both components without their previous separation. The precision of the method for the assay of salbutamol in tablets was 1.0% with an average recovery of 98.8%. In the assay of the two-component preparation, the precision was 1.1%, with average recoveries for salbutamol of 99.3% and for beclomethasone dipropionate of 99.4%.

Influence of single versus multiple actuations on the particle size distribution of beclometasone dipropionate metered-dose inhalers.

The particle size distributions of beclometasone dipropionate delivered from Becotide and Respocort inhalers after single and multiple actuations were investigated using the Andersen Mark II Cascade impactor and the drug was quantified using high performance liquid chromatography. The fine particle mass and the mass median aerodynamic diameter were calculated. An apparent increase in mass median aerodynamic diameter was observed when the number of actuations increased. In addition, the fine particle mass decreased as the number of actuations increased. When performing and analysing cascade impaction study data differences between single versus multiple actuations must be considered. Regulatory guidelines should be amended to stipulate the number of actuations to be loaded into devices used to evaluate the particle size distribution of inhaled aerosol products.

Identification of ten corticosteroids in human hair by liquid chromatography-ionspray mass spectrometry.

This paper describes a screening procedure based upon high-performance liquid chromatography-ionspray mass spectrometry for the identification of ten corticosteroids in human hair: triamcinolone, prednisolone, prednisone, methylprednisolone, cortisone, cortisol, beta- and dexamethasone, flumethasone and beclomethasone. Hair strands were washed in methylene chloride, pulverized in a ball mill and 50 mg of the powdered hair were incubated in 1 ml Soerensen buffer, pH 7.6 for 16 h at 40 degrees C, in presence of 50 ng cortisol-d3 used as internal standard. Purification of the incubation medium was achieved on SPE C18 Isolute extraction columns. The eluates were evaporated to dryness and resuspended in 30 microliters MeOH before analysis by HPLC-IS-MS in positive and negative modes of detection. The validation parameters were found satisfactory for a corticosteroid screening procedure. The correlation coefficient of the calibration curve ranged from 0.939 to 0.997, showing linearity between 0.1 and 10 ng/mg, excepted for beclomethasone which was between 0.2 and 10 ng/mg. Extraction recovery at 4 ng/mg ranged from 43.2 to 85.7%. Repeatability (CV values) at 4 ng/mg ranged from 6.1 to 17.5%. The limits of detection ranged from 0.03 to 0.17 ng/mg for a signal-to-noise ratio of 2. The detection of prednisone and beclomethasone in three hair specimens obtained from forensic and clinical cases have documented corticosteroids incorporation into human hair.

[Effects of mouth wash on the removing beclomethasone dipropionate delivered by pressurized aerosol metered-dose inhaler in the mouth].

Effects of mouth wash (mouth rinsing and gargling) on the removal of drug residues in both mouth and pharynx after the use of pressurized aerosol metered-dose inhaler (MDI) were studied. The concentration of beclomethasone dipropionate (BM) in mouth wash after a splay of Becotide inhaler was measured by the method using HPLC. The total amount of the removed BM was measured by a sum of the concentrations of BM in 4 or 5 times of mouth washes in the following 4 kinds of methods. In method 1, mouth wash was done with 5 times of water change after a splay of MDI on wetted mouth. In method 2, mouth wash was done with 5 times of change water on dried mouth. In method 3, mouth wash was done with 4 times of change saliva on wetted mouth. In these methods, the actual inhalation of BM was not done. In method 4, mouth wash was done with 5 times of change water after a splay and a inhalation on wetted mouth. The mouth wash procedures removed totally 47.9%, 51.1%, 31.3%, and 33.3% of a splayed amount of BM in each method, respectively. It was required for the removal of 90% of the totally recovered BM to do one time of mouth wash in method 1, two times in method 2, three times in method 3, and two times in method 4, respectively. These data suggest that the mouth wash procedure is shown to have prophylactic benefit for candidiasis induced by steroid delivered by MDI.

[Beclometasone Dipropionate Reference Standard (Control 011) of National Institute of Health Sciences].

The raw material of beclometasone dipropionate was examined for the preparation of the "Beclometasone Dipropionate Reference Standard (Control 011)". The analytical data obtained were: melting point, 208.8 degrees C; optical rotation, [alpha]D20 = +91.7 degrees; IR spectrum, same as that of the Beclometasone Dipropionate Reference Standard (Control 865); thin-layer chromatography, one impurity was detected until 40 micrograms; high-performance liquid chromatography, total amount of impurities estimated to be less than 0.5%; loss on drying, 0.6%. Based on the above results, the raw material was authorized as the Beclometasone Dipropionate Reference Standard (Control 011) of the National Institute of Health Sciences.

Development and validation of simultaneous spectrophotometric and TLC-spectrodensitometric methods for determination of beclomethasone dipropionate and salbutamol in combined dosage form.

Spectrophotometric and TLC-spectrodensitometric methods were developed and validated for the simultaneous determination of beclomethasone dipropionate (BEC) and salbutamol (SAL). The spectrophotometric methods include dual wavelength, ratio difference, constant center coupled with a novel method namely, spectrum subtraction and mean centering with mean percentage recoveries and RSD 99.72±1.07 and 99.70±1.12, 100.25±1.12 and 99.89±1.12, 99.66±1.85 and 99.19±1.32, 100.74±1.26 and 101.06±0.90 for BEC and SAL respectively. The TLC-spectrodensitometric method was based on separation of both drugs on TLC aluminum plates of silica gel 60 F254, using benzene: methanol: triethylamine (10:1.5:0.5 v/v/v) as a mobile phase, followed by densitometric measurements of their bands at 230 nm. The mean percentage recoveries and RSD were 99.07±1.25 and 101.35±1.50 for BEC and SAL respectively. The proposed methods were validated according to ICH guidelines and were applied for the simultaneous analysis of the cited drugs in synthetic mixtures and pharmaceutical preparation. The methods were found to be rapid, specific, precise and accurate and can be successfully applied for the routine analysis of BEC and SAL in their pharmaceutical formulation with no need for prior separation. The results obtained were statistically compared to each other and to that of the reported HPLC method. The statistical comparison showed that there is no significant difference regarding both accuracy and precision.

Sensitive and robust methods for simultaneous determination of beclomethasone dipropionate and formoterol fumarate dihydrate in rotacaps.

Fixed dose combination containing beclomethasone dipropionate (BDP) and formoterol fumarate dihydrate (FFD) is used in the treatment of asthma in form of dry powder inhaler. Two methods are described for the simultaneous determination of BDP and FFD in commercial rotacap formulation. The first method was based on HPTLC separation of the two drugs followed by densitometric measurements of their spots at 220 nm. The separation was carried out on Merck HPTLC aluminum sheets precoated with silica gel 60F254 using hexane:ethyl acetate:methanol:formic acid (2.0:2.5:2.0:0.2, v/v/v/v) as mobile phase. The linearity was found to be in the range of 2.4-8.4 µg/spot and 80-280 ng/spot for BDP and FFD, respectively. The second method was based on HPLC separation of the two drugs on the reversed phase Enable HPLC Analytical C18 G 120Å (250 × 4.6 mm, 5 µm) column at ambient temperature using a mobile phase consisting of methanol:acetonitrile:phosphate buffer adjusted to pH 3.6 using orthophosphoric acid (65:25:10, v/v/v). Quantitation was achieved with UV detection at 220 nm based on peak area with linear calibration curves at concentration ranges of 10-200 and 0.3-6.0 µg/mL for BDP and FFD, respectively. Both methods were validated in terms of precision, robustness, recovery and limits of detection and quantitation. The robustness of both methods was assessed using experimental design and results were analyzed by statistical and graphical approaches. Rotacaps formulation containing BDP (200/400 µg) and FFD (6 µg) were successfully quantified using the proposed methods. The proposed methods can be used as sensitive, precise, accurate and robust methods for quantification of BDP and FFD in Rotacaps.