Ultra-performance Liquid Chromatography Method Research Articles

Quality by Design Tool Assessed Ultraperformance Liquid Chromatography Method for the Analysis of Remogliflozin and Teneligliptin in Oral Dosage Form.

The UPLC methodology was used to establish a method for determining the qualitative and quantitative content of teneligliptin and remogliflozin tablets in oral solid dose form, as no simultaneous method was available. The developed liquid chromatography method consists of an X-Bridge C18 100 mm × 3.5 mm, 2.1 mm column with an economical 0.2 mL/min flow rate. A wavelength of 248 nm was used for detection, and the temperature of the column compartment was 30 °C. The method was evaluated using a static tool quality by design after it was validated as per the regulations. The data from validation result in linearity for both analytes with a correlation coefficient of more than 0.999. The accuracy data were found from a minimum of 98.1 to a maximum of 100.9. All of the validation results met the acceptance criteria. The stability of the analytical solutions proved for 24 h at bench and refrigerator temperatures. Studies of force degradation proved the stability indicating the nature of the method. A factorial design was used to evaluate the method performance.

A UPLC method development and validation study of Upadacitinib and its impurities in extended – release oral tablet dosage forms

The primary objective was to develop a concomitant isocratic ultra-performance liquid chromatographic photo-diode array detection method to estimate Upadacitinib and its process-related impurities: impurity-1 and impurity-2. Further validation was conducted and studied for possible degradants under stress environments. All the chemicals and reagents used were of HPLC (acetonitrile, methanol) and analytical grade (trifluoro acetic acid). The ultra-performance liquid chromatography (Agilent 1290 Infinity II LC system) consists of a quaternary pump, a BEH C18 (50×2.1mm, 1.7μ) column, and photo-diode array detector. The method was developed with acetonitrile: methanol: 0.1% v/v trifluoro acetic acid (50:20:30 v/v/v) mobile phase at 0.2mL/min flow rate within a run time of 5.5min The detection was carried at 231.2nm. The respective retention times achieved were 2.289min (Upadacitinib), 0.972min (Upadacitinib impurity-1), and 3.508min (Upadacitinib impurity-2). The optimized method was validated further, and the linearity range was best fit at 15.0-180.0μg/mL for Upadacitinib and 1.0-12.0μg/mL for both Upadacitinib impurity-1 and 2 respectively. The detection and quantification limits were 4.50μg/mL, 15.00μg/mL (Upadacitinib) and 0.30μg/mL, 1.0μg/mL (Upadacitinib impurity-1 and 2). A fast, isocratic, specific, and reproducible ultra-performance liquid chromatographic method was developed and validated for various parameters according to the ICH Q2 (R1) guidelines studies. Stress studies were conducted exposing the sample dilution to various treatments (acid, alkali, peroxide, HPLC water, heat, and UV light). The degradants were well-separated apart from the peaks of the active substance. The stability indicating nature was observed during the degradation. The optimized method can be applied for the separation and estimation of Upadacitinib and its process-related impurities in pharma sector in tablet dosage forms.

Evaluation of the in vitro human skin percutaneous absorption of ketoprofen in topical anhydrous and aqueous gels.

Ketoprofen is a nonsteroidal anti-inflammatory drug used for the treatment of acute and chronic pain associated with inflammatory conditions. This study aims to evaluate the in vitro percutaneous absorption of ketoprofen 10% formulated in proprietary anhydrous and aqueous gels using the Franz skin finite dose model. The anhydrous gel was initially characterized for cytotoxicity using EpiDerm skin tissue model by cell proliferation assay and Western blot analysis. The Ultra Performance Liquid Chromatography method for measuring ketoprofen was validated and the stability of ketoprofen 10% in the anhydrous gel formulation was evaluated at 5°C and 25°C for 181 days. The percutaneous absorption of ketoprofen was determined using donated human skin. The tissue sections were mounted within Franz diffusion cells. A variable finite dose of each ketoprofen formulation in either anhydrous or aqueous gel was applied to the skin sections and receptor solutions were collected at various time points. Cell proliferation assay showed minimal cell death when EpiDerm skin tissue was exposed to the anhydrous gel for 24h; the levels of protein markers of cell proliferation were not affected after 17-h exposure. Ketoprofen was stable in the anhydrous gel when stored at 5°C and 25°C. When compounded in the anhydrous and aqueous gels, ketoprofen had mean flux rate of 2.22 and 2.50μg/cm2 /h, respectively, after 48h. The drug was distributed to the epidermis and dermis sections of the skin. Both the anhydrous and aqueous gels facilitated the percutaneous absorption of ketoprofen without statistically significant differences. The anhydrous gel can be used as a base to facilitate the transdermal delivery of ketoprofen. Although the anhydrous and aqueous gels can deliver a similar amount of ketoprofen, the anhydrous gel (water activity below 0.6) allows for extended default beyond-use-date of compounding preparations.

Development of a Stability Indicating UPLC Method for the Determination of Tirbanibulin in Bulk and Its Pharmaceutical Dosage Form.

The primary goal of this study was to create and validate a simple, precise, sensitive, and accurate ultra-performance liquid chromatography (UPLC) method for estimating tirbanibulin in pure and dosage form. A UPLC technique was developed using a Waters Acquity UPLC Phenyl (100 x 2.1 mm, 1.7 µm) column. The developed technique was validated in accordance with the International Conference on Harmonization (ICH) guidelines. Tirbanibulin was separated chromatographically with high resolution using the mobile phase acetonitrile: buffer (30:70 v/v) at 0.5 mL/min, 5 µL injection volume, and 220 nm wavelength. The validated technique was found to be linear in the 1-15 µg/mL range. The detection and quantification limits for tirbanibulin were 0.03 and 0.1 µg/mL, respectively. The percentage relative standard deviation was less than 2%, demonstrating the precision of the developed technique. Furthermore, the recovery rate was nearly 100%, confirming the accuracy of the method. Minor modifications to the chromatographic conditions demonstrated the robustness of the method. The developed analytical method was precise, simple, reproducible, and sensitive. Consequently, it can be used to determine tirbanibulin.

A NOVEL VALIDATED STABILITY INDICATIVE UPLC METHOD FOR RELUGOLIX FOR THE DETERMINATION OF PROCESS-RELATED AND DEGRADATION IMPURITIES

A NOVEL VALIDATED STABILITY INDICATIVE UPLC METHOD FOR RELUGOLIX FOR THE DETERMINATION OF PROCESS-RELATED AND DEGRADATION IMPURITIES

Development and Validation of the Ultra-Performance Liquid Chromatography Method for Quantification of Belzutifan in Pharmaceutical Dosage Form

Development and Validation of the Ultra-Performance Liquid Chromatography Method for Quantification of Belzutifan in Pharmaceutical Dosage Form

Stability Indicating UPLC Method Development and Validation for the Quantitative Estimation of Pazopanib in Pure form and Marketed Pharmaceutical Dosage form.

An analytical, accurate, precise, specific, efficient and simple Ultra-Performance Liquid Chromatography method has been developed and validated for the determination of Pazopanib in bulk and was applied on marketed Pharmaceutical Dosage form. The mobile phase used for the chromatographic runs consisted of 0.1% OPA Buffer and Acetonitrile in the ratio of 30:70% v/v. The separation was achieved on a BHEL UPLC column using isocratic mode. Pazopanib Drug peak were well separated and were detected by a PDA detector at 256 nm. The developed method was linear at the concentration range 6-14 μg/ml for Pazopanib. The method has been validated according to ICH guidelines with respect to system suitability, specificity, precision, accuracy and robustness. The LOD and LOQ for the Pazopanib were found to be 0.5853 µg/ml and 1.7738µg/ml respectively. The developed method is simple, precise, specific, accurate and rapid, making it suitable for estimation of Pazopanib in bulk and marketed pharmaceutical dosage form dosage form.

Stability Indicating Reverse Phase Ultra Performance Liquid Chromatography Method Development and Validation for Amiloride and Hydrochlorothiazide Tablet in the Presence of Degradation Products and Application to Green Analytical Principles

Amiloride (AML) and hydrochlorothiazide (HCTZ) combination is used alone or with other medicines to treat high blood pressure (hypertension). The present article provides the development and validation of an reverse phase ultra performance liquid chromatography (RP-UPLC) approach for the quantification of AML, HCTZ, and related impurities in pharmaceutical dosage forms. The analytical column used for the separation of impurities is the Waters X bridge C18 3.5 μm (50 mm X 4.6 mm). Used 0.1% formic acid and ethanol as MP in an isocratic mode with 0.3 mL/min as flow rate and 3 μL as injection volume, at 254 nm as detection in UV and 12 minutes as total run time. The samples were prepared specifically to undergo forced degradation, which involved subjecting them to hydrolysis, oxidation, thermal, and photolytic conditions. The technique underwent validation by the principles set forth by the ICH Q2 guidelines. The validation confirmed that the method possesses specificity, linearity, ruggedness, robustness and accuracy. The methodology employed exhibited a linear relationship extending from 10 to 150% for all impurities. The recovery analysis was conducted throughout a range of concentrations, starting from 10 to 150% concentration. The average recovery value was determined to be within acceptable limits. The evidence of degradation and the findings of the verified study suggest that the nature of the subject under investigation is stable. Hence, this approach might be employed within the domains of pharmaceutical research and development and quality control departments. Green analytical chemistry tools are used to assess the method’s greenness and calculated using GAPI, AGREE and ecological-scale and found excellent green of >75%.

Comparing baseline correction algorithms in discriminating brownish soils from five proximity locations based on UPLC and PLS-DA methods.

UPLC data of soil interfere with baseline drifts.BC can improve the quality of the pixel-level UPLC data.MW emerges as the most desired algorithm in improving the quality of UPLC data of soil.

An ultra performance liquid chromatography method for transthyretin variants screening and heart failure assisting diagnosis

BackgroundTransthyretin (TTR) gene mutations are associated with hereditary amyloidosis (ATTR) caused by mutant TTR protein dissociation, misfolding, aggregation, and insoluble fibrils deposition. Herein, we reported a chromatographic approach for quantification and identification of TTR tetramer in human blood serum by ultra performance liquid chromatography (UPLC). MethodsTTR proteins and serum were incubated with a fluorescent TTR tetramer sensor (A2). The A2 sensor specifically reacted with tetrameric TTR and released stoichiometric fluorescence that was detected by fluorescence detector coupled to UPLC. The external standard was used for quantification, the chromatographic peak parameters were used to identification certain mutation types. ResultsUPLC correctly distinguished 18 types of mutant TTR proteins from wild type. The results were consistent with follow-up analysis of two ATTR patients’ blood serum samples. In addition, the tetrameric TTR of 30 heart failure (HF) patients showed strongly correlation (r = −0.63, p < 0.00) with NT-proBNP, a HF clinical biomarker. ConclusionsUPLC method has sufficient accuracy to eliminate the necessity of sequencing for certain types of TTR mutations and allows for facile initial screening of ATTR amyloidosis patients, carriers, and healthy individuals for time-saving and economical purposes. TTR tetramer may serve as a diagnostic biomarker to evaluate the risk of HF diseases.

Development and validation of a stability-indicating ultra-high-performance liquid chromatography method for the estimation of ibrutinib and trace-level quantification of related substances using quality-by-design approach.

A new ultra-high-performance liquid chromatography method was developed using quality-by-design principles for quantifying trace-level impurities of ibrutinib. The method utilized an ACQUITY UPLC BEH C18 column with a mobile phase consisting of equal parts of 0.02 M formic acid in water and 0.02 M formic acid in acetonitrile. The critical method parameters, including mobile phase pH, column temperature, and flow rate, were optimized using the design of experiments. Statistical analysis revealed the impact of these parameters on critical quality attributes. Perturbation and response surface plots illustrated the individual and interactive effects of the parameters. The optimal parameter levels were determined to be pH,2.5; column temperature, 28°C; and flow rate, 0.55 mL/min. Confirmation experiments demonstrated the method's robustness, with the separation of impurities and unknown degradation products within a 5-min runtime. The optimized ultra-performance liquid chromatography method was validated according to ICH guidelines. The method exhibited linear response within the range of 0.025-100 μg/mL for ibrutinib and 0.0187-0.225 μg/mL for impurities (r2 > 0.9995), with limits of detection/limits of quantification of 0.01/0.025 and 0.015/0.0187 for ibrutinib and four impurities, respectively. Recoveries for the drug and impurities ranged from 92.69 to 102.7%, and precision was below 2% and 8% relative standard deviation for ibrutinib and impurities, respectively.

Determination of Clorsulon and its related substances in commercial bulk drug substance batches by a rapid ion-pair UPLC method

Clorsulon is a potent anthelmintic agent and is widely used for the treatment and control of parasites including adult liver flukes in cattle and sheep. A rapid ion-paired reversed phase ultraperformance liquid chromatography (IP-UPLC) method has been developed and validated for determination of Clorsulon and its related substances in bulk drug substance batches of Clorsulon with a short octadecyl column. Analytes were eluted by a gradient elution on a Acquity UPLC® BEH C18 column (50 mm × 2.1 mm i.d., 1.7 µm particle size). Column temperature was maintained at 55 °C and all analytes were monitored by UV detection at 268 nm. Mobile phase-A constitutes 3 mM tetrabutylammonium hydroxide in H2O and mobile phase-B constitutes acetonitrile/methanol (50/50, v/v), respectively. All analytes of interest were adequately separated within 5 min. The analytes were quantitated against an external reference standard of Clorsulon. The chemical structures of degradation products of Clorsulon were proposed based on two-dimensional UPLC-MS data. The IP-UPLC method has been successfully validated and demonstrated to be accurate, sensitive, robust, and specific.

A validated green UPLC method for the quantification of synthetic pharmaceutical adulterants in the extract of the aphrodisiac herbal plant ashwagandha (Withania somnifera) and in spiked human plasma

In this work, an ultra-performance liquid chromatographic method was developed and subjected to bioanalytical method validation for screening the root extract of Ashwagandha. The method was also extended for simultaneous determination of Sildenafil, Vardenafil and Avanafil in plant root extract and in human plasma samples to detect the possible counterfeiting of the plant's pharmaceutical products with other synthetic drugs that might endanger the life of their consumers, particularly elderly people, and those with chronic diseases. The method was developed using BDS Hypersil TM C18 column (250 × 4.6 mm and 3 μm particle size) as a stationary phase and a mobile phase consisting of acidified water and acetonitrile in a gradient elution program with a flow rate of 1.00 mL min 1. In the plant extract, the proposed method was found to be linear in the range 1.00–200.0 μg mL−1 for the Vardenafil and Avanafil and 5.00–200.0 μg mL−1 for Sildenafil with a recovery percentage in range of 98.18–101.07 % for the three drugs. Intra-day and inter-day precision ranges were 0.088–1.219 and 0.151–1.313, respectively. The limit of detection and the limit of quantification for Vardenafil and Avanafil were 0.166 μg mL−1 and 0.500 μg mL−1, respectively. Whereas, for Sildenafil 0.333 μg mL−1 and 1.000 μg mL−1. The ICH validation parameters were also demonstrated. Bioanalytical method validation was established following US-FDA recommendations and the method showed good linearity in the range 1.00–200.00 μg mL −1 for Vardenafil and Avanafil, and 5.00–200.00 μg mL −1 for Sildenafil. The results revealed high accuracy with % recovery ranging from 91.05 to 98.91 % for the three drugs. The intra-day and inter-day precision were also conducted, in terms of % relative standard deviation, and ranged from 0.119 to 4.037 % and from 0.223 to 5.062 %, respectively. Furthermore, other validation parameters were evaluated including extraction recovery and stability of the analytes throughout samples preparation and storage for both short- and long-term. The method greenness was evaluated using NEMI, GAPI, AGREE and analytical eco-scale.

Development and Validation of a Stability-indicating UPLC-DAD Method for the Simultaneous Determination of Ivermectin and Praziquantel in Pharmaceutical Tablets and Dissolution Media.

Currently, there is no single rapid and accurate stability-indicating quantitative method that can simultaneously determine both ivermectin and praziquantel and their related compounds. Thus, the goal of this research is to develop and validate a new rapid, accurate, and stability-indicating ultra-performance liquid chromatography (UPLC) method. The method uses a water, acetonitrile, and methanol gradient. The chromatographic separation was achieved on a C18 (1.7 μm, 2.1 × 50 mm) column with a flow rate of 0.7 mL/min, and the column temperature was maintained at 40°C. Analytes are detected at 245 nm. The method was validated in accordance with ICH Q2R1 guidelines. The linearity (R2) was >0.9987 and 0.9997 for praziquantel and ivermectin, respectively. The corresponding accuracy ranged between 98.0 and 102.0%. Intermediate precision (assessed as inter-day precision) was determined by calculating the cumulative %CV of eighteen assay preparations and was less than 2.0% for both praziquantel and ivermectin. The specificity of the method was shown by the resolution of the two active pharmaceutical ingredients (APIs) from any interfering excipients, impurities, or degradation products. The limit of detection and quantitation for ivermectin was 26.80 ng/mL and 81.22 ng/mL, respectively. The limit of detection and quantitation for praziquantel was 1.39 μg/mL and 4.22 μg/mL, respectively. The robustness study proved that method performance is stable against small variations in sample processing parameters (shaking, sonication time, and acetonitrile % in solvent solution) and also against small variations in the initial % of mobile phase components and gradient slope. Using ICH Q2R2 criteria, the method was demonstrated to be specific, accurate, stability indicating, and robust to small variations of chromatographic variables.

New validated Ultra High Performance Liquid Chromatographic method for Estimation of Quercetin

Background: A new Ultra performance liquid chromatographic analytical method was developed and validated for estimation of Quercetin in bulk powder. The reverse phase chromatographic elution using UPLC was carried out in gradient mode on C-18 column (Phenomenex Luna 5 µm, 150 mm X 4.6 mm) as stationary phase utilizing a mobile phase composed of Acetonitrile: 0.1 % formic acid buffer (50:50 v/v) with a flow rate of 1 ml/min and injection volume of 10µl. The analysis was performed at temperature of 400C and detection of eluent was carried out using photo diode array at 371 nm. The newly developed Ultra performance liquid chromatography method was validated in terms of linearity and range, system suitability, specificity, precision, sensitivity, robustness, ruggedness and accuracy as per ICH guidelines. Results: The retention time of quercetin was found to be at 3.9 min with total run time of analysis is 7 minutes. The linearity was observed between the concentration ranges from 0.5 to 16µg/ml with correlation coefficient 0.999. The precision assays values were found to be less than 2% for both the drugs. The Limit of Detection and Limit of Quantification were 0.47µg/ml and 1.44µg/ml for Quercetin. The mean percentage recovery values were found to be within the range of 90-110 %. Conclusion: The proposed method was found to be simple, specific, precise, sensitive, robust, rugged and accurate and can be used for routine quality control analysis of Quercetin.

Development and Validation of a New Reverse Phase Ultra Performance Liquid Chromatography Method for the Estimation of Related Substances of the Anticancer Drug Zanubrutinib and Characterization of its Degradants Using Liquid Chromatography Mass Spectrometry

Development and Validation of a New Reverse Phase Ultra Performance Liquid Chromatography Method for the Estimation of Related Substances of the Anticancer Drug Zanubrutinib and Characterization of its Degradants Using Liquid Chromatography Mass Spectrometry

Comparison of chemical constituents in Artemisiae Argyi Folium from different Dao-di producing areas based on UPLC and HS-GC-MS

This study aims to compare the chemical constituents in 24 batches of Artemisiae Argyi Folium samples collected from three different Dao-di producing areas(Anguo in Hebei, Nanyang in Henan, and Qichun in Hubei). An ultra-performance liquid chromatography(UPLC) method was established to determine the content of 13 nonvolatile components, and headspace-gas chromatography-mass spectrometry(HS-GC-MS) was employed for qualitative analysis and comparison of the volatile components. The content of phenolic acids in Artemisiae Argyi Folium was higher than that of flavonoids, and the content of nonvolatile components showed no significant differences among the samples from the three Dao-di producing areas. A total of 40 volatile components were identified, and the relative content of volatile components in Artemisiae Argyi Folium was significantly different among the samples from different Dao-di producing areas. The principal component analysis and partial least squares discriminant analysis identified 8 volatile components as the potential markers for discrimination of Artemisiae Argyi Folium samples from different Dao-di producing areas. This study revealed the differences in the chemical composition of Artemisiae Argyi Folium samples from three different Dao-di producing areas, providing analytical methods and a scientific basis for the discrimination and quality evaluation of Artemisia Argyi Folium in different Dao-di producing areas.

A Novel Approach to AQbD-Assisted Green and Robust UPLC Method for the Determination of Selected Aminoquinoline Antimalarial Drugs: Assessment of Green Method

Greening analytical procedures have gained popularity in the area of pharmaceutical industries in order to safeguard the environment. In this study, an innovative methodology by integrating analytical quality-by-design (AQbD) along with Green Chemistry concepts with a systematic approach has been used to develop an environmentally-friendly and more robust ultra performance liquid chromatography (UPLC) method for the determination of three selected aminoquinoline antimalarial drugs namely amodiaquine hydrochloride (AMD), chloroquine phosphate (CHQ) and piperaquine phosphate (PPQ). An environmentally benign solvent ethanol has been chosen as an alternative to the commonly used acetonitrile and methanol, which are widely used. Based on the comprehensive risk analysis, the critical method parameters (CMP’s) that affect the critical method attributes (CMA’s) have been identified and the final method parameters were further optimized with the help of a full factorial design and the design of experiments (DOE), established the “design space”(DS) and identified the “method-operable-design-region (MODR) for the developed method. Achieved the separation with the use of an Acquity- UPLC BEH Shield RP-18 (100 mm × 2.1 mm), 1.7 μm column by using an isocratic elution of mobile phase containing 0.1% trifluoroacetic acid in water, ethanol in a mixture of 90:10% v/v and maintained a flow rate of 0.2 mL min-1 and the column oven temperature was 40 ºC and the UV detection wavelength of 225 nm was employed. A statistical tool Analysis of Variance (ANNOVA) has been used to assess the model’s statistical significance and found that the model’s p-values is &lt; 0.00005 and its lack of fi is &gt; 0.05 respectively, which demonstrates that the selected model is the most appropriate predictive tool for the analyzed responses R1 &amp; R2 (i.e. peak resolutions R1 &amp; R2). The proposed method’s linearity, accuracy &amp; precision studies has been successfully validated in compliance with USP &lt;1225&gt; and ICH Q2 (R1) guidelines. Further assessed the proposed method’s greenness using various green evaluation tools such as: “complex green analytical procedure index”-cGAPI, “analytical greenness”-AGREE, “analytical method green score”-AMGS and found acceptable results. This novel AQbD-assisted eco-friendly analytical method for the quantification of AMD, CHQ &amp; PPQ in bulk drugs (APIs) or finished dosage forms is simple, rapid, precise and accurate, robust and environmentally benign.

A new Wastewater-Based Epidemiology workflow to estimate community wide non-communicable disease prevalence using pharmaceutical proxy data

This manuscript introduces a new wastewater-based epidemiology workflow for estimation of non-communicable diseases (NCDs) prevalence by using wastewater-based epidemiology (WBE) and pharmaceuticals/their metabolites as proxies for NCDs prevalence. 83 targets were selected (54 parent pharmaceuticals and 29 metabolites). Three critical aspects were tested: (i) Solid-Phase Extraction - Ultra-Performance Liquid Chromatography and Tandem Mass Spectrometry (SPE-UHPLC-MS/MS) method performance, (ii) biomarker stability under variable storage conditions (during sampling and long-term storage) and (iii) accounting for human metabolism in WBE back-calculations. High stability of most analytes was observed under tested storage conditions. A few exceptions include diazepam, dihydroketoprofen and 5-hydroxy-lansoprazole. Analyte recoveries varied between 75% and 125% for most analytes. MDLs ranged from 0.2 ng L-1 to 5.6 ng L-1, while MQLs from 0.2 ng L-1 to 16.8 ng L-1. The overall average method accuracy and precision were: 99.5% and 4.0% respectively. A fully validated method was tested using community wastewater in the Southwest of England to estimate pharmaceutical usage, test metabolism correction factors established and compare results with prescription data. The new WBE method for NCD approximation allowed for the estimation of the daily usage/intake of 69 NCD targets with a standardized approach and a consistent reporting format.

Design of Experiment-Based Green UPLC-DAD Method for the Simultaneous Determination of Indacaterol, Glycopyrronium and Mometasone in their Combined Dosage Form and Spiked Human Plasma.

Abstract Indacaterol, is an ultra-long-acting β2 agonist, glycopyrronium is a long-acting muscarinic-antagonist and mometasone is a synthetic corticosteroid. They were used recently in combination for the treatment of severe asthma symptoms and chronic obstructive pulmonary disease. In this work, it was the first time to develop a green and environment friendly ultra-performance liquid chromatographic method using design expert program for the analysis of the three drugs in their combined dosage form. Also, the method was bioanalytically validated for the analysis of the three drugs in spiked human plasma samples. The method was linear in range from 0.50 to 100.0 μg mL−1 for indacaterol and mometasone and from 1.0 to 150.0 μg mL−1 for glycopyrronium. It showed high accuracy where, the % recovery for indacaterol, glycopyrronium and mometasone in plasma were ranged from 94.27 to 97.86%, 96.43 to 98.75% and 96.86 to 98.43%, respectively. Also, it was precise where, the % relative standard deviation for the inter-day precision was ranged from 2.571 to 3.484%, 3.180 to 4.123% and 3.150 to 3.984% and the intra-day precision was ranged from 2.351 to 3.125%, 2.512 to 3.544% and 2.961 to 3.983% for indacaterol, glycopyrronium and mometasone, respectively. The limit of detection and the limit of quantification for indacaterol and mometasone were 0.03 and 0.10 μg mL−1 while for glycopyrronium, they were 0.16 and 0.50 μg mL−1. Highlights