Stability-indicating High-performance Liquid Chromatography Method Research Articles

Stability-Indicating HPLC Method for Determination of Ibuprofen and Famotidine Degradation Products.

A new stability-indicating high-performance liquid chromatographic method for the quantitative determination of ibuprofen and famotidine degradation products in combined pharmaceutical products was developed and validated. The current aim of this study is to develop a rapid, accurate and robust analytical stability indicating impurity method that can separate ibuprofen, famotidine and their related impurities by using a reversed-phase high-performance liquid chromatography. A Zorbax SB-Phenyl column (4.6 × 150mm2, 5-μm particle size) with mobile phase containing phosphate buffer solution with a pH value of 3.0 and acetonitrile was used. The flow rate was 0.8mL/min and the analytes were detected by UV detector at 265nm. The retention times of ibuprofen and famotidine were 18.43 and 5.14min, respectively. This method was validated to confirm specificity, linearity, sensitivity (limit of detection and limit of quantitation), precision, accuracy, robustness and sample stability according to the International Conference on Harmonization guidelines. Studies have been completed and reported with two active substances in the combined dosage form and seven impurities in total. There is no method in the literature that simultaneously distinguishes and quantitatively analyzes both active substances and degradation products.

A quality-by-design evaluated liquid chromatography method development and validation for the separation and quantification of nitroxoline and its impurities.

A novel, isocratic, sensitive, stability-indicating high-performance liquid chromatography method was developed for the separation and quantification of related substances in nitroxoline (NTL). The chromatographic separation has been achieved on Inertsil ODS-3V, (250 × 4.6mm, 5μm) at 240nm using ethylenediamine tetraacetic acid buffer and methanol in the ratio of 60:40 v/v as mobile phase. The performance of the method has been checked as per the International Conference on Harmonization guidelines for specificity, linearity, accuracy, precision, and robustness. Regression analysis showed a correlation coefficient value greater than 0.99 for NTL and its three impurities. The detection limit of impurities was in the range of 0.01% (0.05μg/mL)-0.22% (1.1μg/mL) indicating the sensitivity of the newly developed method. The accuracy of the method was established based on the recovery obtained between 94.7% and 104.1% for all the impurities. The percentage relative standard deviation obtained for the repeatability was less than 4.0% at the specification level for all impurities. Forced degradation was performed to establish the stability-indicating nature of the method and to know about the degradation products, the quality of a drug substance changes with time under the influence of stress conditions. Thus, the proposed method was validated and found to be specific, sensitive, linear, accurate, precise, reproducible, and beneficial for routine usage.

Separation and quantitative estimation of stereo-selective enantiomers of montelukast in pharmaceutical drug substance and tablets dosage forms by using stability-indicating normal phase-HPLC method.

Montelukast sodium (MLS) is a leukotriene receptor antagonist that relieves asthma, bronchospasm, allergic rhinitis, and urticaria. A simple, robust, and stability-indicating normal phase high-performance liquid chromatography method was developed to separate and quantitatively estimate the S-enantiomer of MLS. The chiral separation was achieved using USP L51 packing material along with a mobile phase consisting of a solvent mixture (n-hexane, ethanol, and propionic acid), a flow rate of 1.0mL/min, a detection wavelength of 284 nm, a column temperature of 30°C and an injection volume of 20 μL. The enantiomers peaks were well separated from the peaks of the placebo, diluting solvent, MLS, and its known impurities with a resolution of more than 2.2 and with no interference. Accuracy and linearity were studied in a range of 0.36-3.597 μg/mL (0.03%-0.30%), with good recoveries between 92.5% and 96.8% and a linear regression coefficient above 0.996. The suggested chiral chromatography method is being considered as an alternative and equivalent method to the United States Pharmacopeia and European Pharmacopeia monographs. The developed method was effectively employed for the study of release and stability samples of MLS. This HPLC method is also capable of separating and estimating the stereo-selective isomers (R- and S-enantiomers) of sulfoxide impurity of MLS in pharmaceutical medicine.

Environmentally friendly stability-indicating HPLC method for the determination of isotretinoin in commercial products and solubility samples

In this study, an environmentally friendly “high-performance liquid chromatography (HPLC)” assay to quantify isotretinoin (ITN) in commercial products and solubility samples is designed and verified. A Nucleodur reverse-phase C18 column was used as the stationary phase to identify ITN. The ecologically friendly mobile phase was composed of ethyl acetate and ethanol (50:50 v/v), and it was delivered at a flow rate of 1.0 mL/min. ITN was measured at 354 nm in wavelength. The current HPLC method had a determination coefficient of 0.9994 and was linear in the 0.2–80 μg/g range. The current protocol for ITN measurement was also rapid (retention time = 2.78 min), accurate (%recoveries = 98.60–101.52), precise (% uncertainties = 0.71–0.98), and sensitive. According to the AGREE methodology, the current procedure received an outstanding greenness profile with an AGREE score of 0.76. By determining ITN in commercial products and solubility samples, the applicability of the current approach was proven. ITN was discovered to be present in 98.43% and 100.84%, respectively, of commercial capsule brands A and B. The ITN's solubility in numerous eco-friendly solvents was successfully measured. Under different stress conditions, the current approach was able to distinguish between its degradation products, demonstrating its stability-indicating characteristics. These findings indicated that ITN in procured capsules and solubility samples might be regularly tested by the suggested approach.

Green micellar stability-indicating high-performance liquid chromatography method for determination of rupatadine fumarate in the presence of its main impurity desloratadine: Oxidative degradation kinetics study.

A green micellar stability-indicating high-performance liquid chromatography method was developed for rupatadine fumarate determination in existence with its main impurity desloratadine. Separation was attained using Hypersil ODS column (150×4.6mm, 5μm), the micellar mobile phase consisted of 0.13M sodium dodecyl sulfate, 0.1M disodium hydrogen phosphate adjusted by phosphoric acid to pH 2.8 and 10% n-butanol. The column was maintained at 45◦ C and detection was carried out at 267nm. A linear response was achieved over the range of 2-160μg/ml for rupatadine and 0.4-8μg/ml for desloratadine. The method was applied for rupatadine determination in alergoliber tablets and alergoliber syrup without the interference of methyl paraben and propyl paraben present as main excipients. Rupatadine fumarate revealed pronounced susceptibility to oxidation; further study of oxidative degradation kinetics was carried out. Rupatadine was found to follow pseudo-first-order kinetics when exposed to 10% H2 O2 at 60 and 80°C and the activation energy was found to be 15.69Kcal/mol. At a lower temperature (40°C), degradation kinetics regression was best fitted as a polynomial quadratic relationship, thus rupatadine oxidation at a lower temperature tends to adopt a second-order kinetics rate. Oxidative degradation product structure was revealed using infrared and found to be rupatadine N-oxide at all temperature values.

A New Stability-Indicating RP-HPLC Method for Simultaneous Quantification of Diacerein and Aceclofenac in Novel Nanoemulgel Formulation and Commercial Tablet Dosage Form in the Presence of their Major Degradation Products Using DoE Approach.

The present study aimed to develop a simple, robust, sensitive and effective stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous quantification of diacerein (DCN) and aceclofenac (ACE) in novel nanoemulgel formulation and commercial tablets in the presence of their main degradation product: rhein (RH) and diclofenac sodium (DLS), respectively. A fractional factorial design was used to screen the crucial independent factors, whereas a central composite design was used for the optimization of the chromatographic conditions. The separation was carried out on Phenomenex C18 column (5μm, 250 × 4.6mm), using a mobile phase consisting of phosphate buffer pH3 (0.1% v/v orthophosphoric acid) and acetonitrile (40:60v/v) at a flow rate of 1mL/min with detection at 264nm. The analytes were exposed to a variety of stress conditions, including heat, alkali, acid, oxidation, photochemical, humidity and hydrolysis. DCN, ACE, RH and DLS were found to have retention times of 4.32 ± 0.15, 5.77 ± 0.07, 8.28 ± 0.20 and 9.10 ± 0.18min, respectively. The percent recovery for all four analytes was found to be between 98 and 102, and the procedure was discovered to be linear in the range of 0.1-64μg/mL with R2 value > 0.999. The established method was validated as per ICH guidelines and successfully used to assay DCN and ACE in their combined marketed tablet dosage form and developed nanoemulgel formulation.

Stability indicating HPLC method development and validation of Fostemsavir in bulk and marketed formulations by implementing QbD approach

Background: Achieving a predictable degree of quality with intended and planned specifications is known as "quality by design." QbD (Quality-by-Design) is an alternative to conventional method development that places more attention on identifying and mitigating potential risks. Component of the Quality-by-Design methodology involves conducting a series of experiments to learn how various factors, including the dependant variables, affect the answers of interest. Here, we use a QbD (Quality-by-Design) loom to detail the creation and verification of a stability-indicating high-performance liquid chromatography (HPLC) method for Fostemsavir in both bulk and finished-goods forms.
 Results:
 In this work, we present a workable experimental design for optimising the RP-HPLC separation technique by identifying the optimum mobile phase concentration and flow rate. Below are the ideal chromatographic conditions as calculated by Design Expert version 13.0: Mobile phase: 80 parts acetonitrile to 20 parts formic acid (v/v), flow rate: 0.8 millilitres per minute, retention time: 3.24 minutes, column dimensions: GIST C18 (250 mm × 4.6 mm × 5.0 μm).
 Here we propose a practical experimental layout for determining the optimal mobile phase concentration and flow rate for the RP-HPLC separation technique. Using Design Expert version 13.0, the optimum chromatographic conditions were determined to be as follows: Shim-pack GIST C18 (250 mm 4.6 mm, 5.0 μ), mobile phase acetonitrile to 1% formic acid (80:20, v/v), flow rate 0.8 ml/min, and retention period 3.24 min.
 At a detection wavelength of 266 nm, it was discovered that the devised technique was linear over a concentration range of 50-90 μg/ml (r2 = 0.997). Test parameters for the system's appropriateness were determined to be 1.124 for the tailing factor and 9480 for the theoretical plates. Intraday RSD was found to range from 0.70 to 0.94, whereas interday RSD was found to range from 0.55 to 0.95 percent. Values for robustness were under 2%. The solution stability % RSD was calculated to be 0.83. The result of the assay was 100.05 percent. The created methodologies were used to studies of forced degradation, and the stressed materials were analysed. The parameters used to validate the procedure fell within the acceptable range recommended by ICH.
 Conclusion: Using Design Expert 13.0, we created a central composite design experiment that illustrates the relationships between mobile phase and flow rate across three levels, with retention duration, tailing factor, as well as theoretical plates as the responses of interest. By this work, we gain insight into the variables that affect chromatographic separation and strengthen our conviction that the HPLC method we've devised will serve our needs. Quantitative method development was applied to improve comprehension of multi-tiered method variables.

Development and validation of a stability-indicating RP-HPLC method for the determination of fifteen impurities in rivaroxaban

A simple and stability-indicating reverse phase high-performance liquid chromatographic (RP-HPLC) method for the determination of rivaroxaban (RIX) and its related substances was developed. Fifteen impurities of RIX, including three unreported isomers, were identified, synthesized, purified, and confirmed using MS, 1H NMR, 13C NMR, and HSQC spectral methods. This new method offered baseline separation for all monitored impurities, and was fast and reliable when compared to the European Pharmacopoeia method. Optimum separation for RIX and its related impurities was achieved on an octyldecyl silica column (YMC Core C18, 4.6 ×100 mm, 2.7 µm) by using a gradient HPLC method in 38 min. The final method was validated with respect to precision, LOD and LOQ, linearity, accuracy, and robustness. This developed method was suitable for routine quality control and drug analysis of RIX active substance.

Analytical Purity Determinations of Universal Food-Spice Curcuma longa through a QbD Validated HPLC Approach with Critical Parametric Predictors and Operable-Design’s Monte Carlo Simulations: Analysis of Extracts, Forced-Degradants, and Capsules and Tablets-Based Pharmaceutical Dosage Forms

Applications of analytical quality by design (QbD) approach for developing HPLC (High Performance Liquid Chromatography) methods for food components assays, and separations of complex natural product mixtures, are still limited. The current study developed and validated, for the first time, a stability-indicating HPLC method for simultaneous determinations of curcuminoids in Curcuma longa extracts, tablets, capsules, and curcuminoids' forced degradants under different experimental conditions. Towards separation strategy, critical method parameters (CMPs) were defined as the mobile phase solvents' percent-ratio, the pH of the mobile phase, and the stationary-phase column temperature, while the peaks resolution, retention time, and the number of theoretical plates were recognized as the critical method attributes (CMAs). Factorial experimental designs were used for method development, validation, and robustness evaluation of the procedure. The Monte Carlo simulation evaluated the developing method's operability, and that ensured the concurrent detections of curcuminoids in natural extracts, commercial-grade pharmaceutical dosage-forms, and the forced degradants of the curcuminoids in a single mixture. The optimum separations were accomplished using the mobile phase, consisting of an acetonitrile-phosphate buffer (54:46 v/v, 0.1 mM) with 1.0 mL/min flow rate, 33 °C column temperature, and 385 nm wavelength for UV (Ultra Violet) spectral detections. The method is specific, linear (R2 ≥ 0.999), precise (% RSD < 1.67%), and accurate (% recovery 98.76-99.89%), with LOD (Limit of Detection) and LOQ (Limit of Quantitation) at 0.024 and 0.075 µg/mL for the curcumin, 0.0105 µg/mL and 0.319 µg/mL for demethoxycurcumin, and 0.335 µg/mL and 1.015 µg/mL for the bisdemethoxycurcumin, respectively. The method is compatible, robust, precise, reproducible, and accurately quantifies the composition of the analyte mixture. It exemplifies the use of the QbD approach in acquiring design details for developing an improved analytical detection and quantification method.

Inclusion Complex of Clomiphene Citrate with Hydroxypropyl-β-Cyclodextrin for Intravenous Injection: Formulation and Stability Studies.

Clomiphene citrate is the first-line treatment for women with abnormal or failed ovulation. Currently, it is available as oral tablets, and the parenteral formulation does not exist. In this study, we prepared clomiphene citrate-hydroxypropyl-β-cyclodextrin inclusion complex for its use in intravenous injection. The inclusion complex was characterized in the liquid state (phase solubility) and solid state by differential scanning calorimetry, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy analyses. The sterile intravenous injection containing 0.5% clomiphene citrate was prepared and characterized for its physical properties, assay, pH, and osmolality. A stability-indicating high-performance liquid chromatography (HPLC) method for the injection was developed. The HPLC method was validated for the assay, linearity, precision and repeatability, benchtop stability, and forced degradation to elute clomiphene isomers from the degradation products. The injection was packed in sterile 10-ml glass vials with butyl rubber stoppers and stored at 40°C, room temperature, and 4°C. The samples at 0, 0.5, 1, 2, 3, and 6months were analyzed for clarity, pH, osmolality, and drug assay. The HPLC method was linear (R2 = 0.9999), precise (0.86% relative standard deviation), and stability indicating. The stability data at the accelerated (40°C) storage condition for 6months showed satisfactory results: the drug assay in the injection was between 90 and 105%, the injection remained clear, pH was between 4.0 and 4.4, and osmolality was between 270 and 350mOsm. The stability data suggests that the product is stable and meets the given analytical specifications.

Development and Validation of Stability-indicating High-Performance Liquid Chromatography Method for Estimation of Organic Impurities of Carvedilol from Bulk and its Dosage Form

Development and Validation of Stability-indicating High-Performance Liquid Chromatography Method for Estimation of Organic Impurities of Carvedilol from Bulk and its Dosage Form

Determination of Firocoxib and Its Related Substances in Bulk Drug Substance Batches of Firocoxib by a High-Speed Reversed-Phase HPLC Method With a Short Fused-Core Biphenyl Column.

Firocoxib is a nonsteroidal anti-inflammatory drug. It provides control of postoperative pain and inflammation associated with soft tissue and orthopedic surgery in dogs, and control of pain and inflammation associated with osteoarthritis in horses. A high-speed stability-indicating reversed-phase high-performance liquid chromatography method was developed to determine firocoxib and its related substances in bulk batches of firocoxib drug substance. Firocoxib was dissolved in neat acetonitrile (ACN) and analyzed on a short HALO (fused-core) biphenyl column (30 × 4.6mm i.d., 2.7-μm particle size) at flow rate of 2.5mL/min. Column temperature was maintained at 50°C. Mobile phase A is composed of 0.1% of H3PO4 in water and mobile phase B is composed of ACN. Analytes were detected with UV detection at 240nm and quantitated against an external reference standard. Firocoxib and its related compounds were adequately separated within 4min by a gradient elution. The method was validated for specificity, linearity, accuracy, precision and robustness according to method validation guidelines described in The International Conference on Harmonization. The validation data demonstrated that this method is sensitive, accurate, robust, specific and stability-indicating.

Development and Validation of Stability-Indicating High-Performance Liquid Chromatography Method for Estimation of Lacosamide in Bulk and Its Pharmaceutical Dosage Form.

A simple, specific, accurate and stability-indicating reversed-phase high-performance liquid chromatographic method was developed for the determination of lacosamide, using C18 column and a mobile phase composed of phosphate buffer (pH4.0):acetonitrile (40:60v/v). The retention time of lacosamide was found to be 2.7min. Linearity was established for lacosamide in the range of 10-50μg/mL. The percentage recovery of lacosamide was found to be in the range of 97.37-99.20%. The drug was subjected to acid, alkali, oxidation, dry heat and photolytic degradation. The degradation studies indicated condition was well resolved from the pure drug with significant differences in their retention time values. This method can be successfully employed for quantitative analysis of lacosamide in bulk drug and formulation.

Degradation pathways and impurity profiling of the anticancer drug apalutamide by HPLC and LC-MS/MS and separation of impurities using Design of Experiments.

Apalutamide, an androgen receptor inhibitor, is used to treat prostate cancer. A stability-indicating high-performance liquid chromatography method was developed for the estimation of assay and organic impurities of apalutamide in drug substance and in tablet dosages using Design of Experiments. The chromatographic separation was achieved within 30 min using Atlantis dC18 , 100 × 4.6mm, 3.0 μm and the binary gradient program (10 mm KH2 PO4 , pH3.5; acetonitrile). The detection wavelength, flow rate, column temperature and injection volume used were 270 nm, 1.0 ml/min, 45°C and 10 μl, respectively. The interaction of independent variables (pH, column temperature and flow rate) and their influences on HPLC parameters were studied using a central composite design, and then the peak separation and elution behaviors between apalutamide and its seven impurities were determined. The method validation was performed for linearity, detection limit, quantitation limit, accuracy, precision and robustness as per the International Conference on Harmonization. A high-quality recovery with good precision (91.7-106.0%) and correlations (r2 > 0.997) within a linear range of 0.12-2.24 μg/ml (0.05-0.3%, w/w) were achieved consistently for assay and organic impurities of apalutamide. The stability-indicating characteristics of the proposed method were assessed through forced degradation and mass balance studies. An effort was made to figure out the chemical structures of newly formed degradation products (DP1-DP5) using LC-MS/MS.

Compatibility and stability of methylprednisolone sodium succinate and granisetron hydrochloride in 0.9% sodium chloride solution.

A combination of methylprednisolone sodium succinate (MSS) and granisetron hydrochloride (GH) is generally devoted to treating the chemotherapy-induced nausea and vomiting. To date, none of these novel mixtures have been commercially available. The present study was aimed at investigating physical and chemical compatibility and stability of a combination of MSS with GH in 0.9% sodium chloride injection for 72 hours at 4°C and 25°C. A mixture of MSS (0.4-0.8 mg/mL) with GH (0.03 mg/mL) was prepared and stored in both polyvinyl chloride bags and glass bottles using 0.9% sodium chloride injection as a diluent. The study was performed using a validated and stability-indicating high-performance liquid chromatography method. The physical compatibility was assessed by a spectrometer. Furthermore, the pH measurement of each sample was measured electronically. All test solutions stored at 4°C or 25°C had a no >2% loss of the initial concentration throughout the 72-hour study period. All solutions remained clear and colorless throughout the study and were without precipitation or turbidity in any of the batches. The drug mixtures of MSS (0.4-0.8 mg/mL) and GH (0.03 mg/mL) in 0.9% sodium chloride injections were physically and chemically stable for at least 72 hours when stored at 4°C or 25°C in polyvinyl chloride bags or glass bottles.

Stability evaluation of compounded hydroxyurea 100 mg/mL oral liquids using a novel analytical method involving chemical derivatization.

This study assessed the stability of six extemporaneously compounded hydroxyurea oral liquids stored at room temperature. Hydroxyurea oral liquids (100 mg/mL) were prepared using three different mixing methods (mortar, mixer or QuartetRx) from either bulk powder, capsule content, or whole capsules. Two brands of capsules were tested in this study. All formulations were stored at room temperature (25°C / 60% RH) in amber plastic bottles for 90 days and amber plastic syringes for 14 days. Physical stability was assessed visually, while chemical stability was evaluated using a stability-indicating high-performance liquid chromatography method. Chemical derivatization with xanthydrol allowed the retention of hydroxyurea on a reverse-phase column. At least 93.9% and 97.0% of the initial concentration of hydroxyurea remained after 90 days in bottles and 14 days in syringes, respectively. There were no visual changes in formulations over the study period. Changes in pH up to 1.6 units were observed after 90 days of storage and were explained most likely by an ammonium generating degradation pathway. Ammonium was quantified and remained within safe levels in each HU 100 mg/mL oral preparations. Hydroxyurea oral liquids were all stable for 90 days in amber plastic bottles and 14 days in amber plastic syringes.

Physicochemical and Microbiological Stability of Two Oral Solutions of Methadone Hydrochloride 10 mg/mL.

In this article, we studied physicochemical and microbiological stability and determined the beyond-use date of two oral solutions of methadone in three storage conditions. For this, two oral solutions of methadone (10 mg/mL) were prepared, with and without parabens, as preservatives. They were packed in amber glass vials kept unopened until the day of the test, and in a multi-dose umber glass bottle opened daily. They were stored at 5 ± 3 °C, 25 ± 2 °C and 40 ± 2 °C. pH, clarity, and organoleptic characteristics were obtained. A stability-indicating high-performance liquid chromatography method was used to determine methadone. Microbiological quality was studied and antimicrobial effectiveness testing was also determined following European Pharmacopoeia guidelines. Samples were analyzed at days 0, 7, 14, 21, 28, 42, 56, 70, and 91 in triplicate. After 91 days of storage, pH remained stable at about 6.5–7 in the two solutions, ensuring no risk of methadone precipitation. The organoleptic characteristics remained stable (colorless, odorless, and bitter taste). The absence of particles was confirmed. No differences were found with the use of preservatives. Methadone concentration remained within 95–105% in all samples. No microbial growth was observed. Hence, the two oral methadone solutions were physically and microbiologically stable at 5 ± 3 °C, 25 ± 2 °C, and 40 ± 2 °C for 91 days in closed and opened amber glass bottles.

Study of erdafitinib stress degradation behavior using HPLC-UV analysis and multistage fragmentation ion trap mass spectrometry

Erdafitinib is a fibroblast growth factor receptor inhibitor, which has been recently approved for the treatment of urothelial carcinoma. The stress degradation behavior of erdafitinib was examined using a validated stability-indicating high-performance liquid chromatography method. The study was extended to investigate the fragmentation pathways of erdafitinib using ion trap mass spectrometry. Six degradation products and an impurity were characterized depending on their multistage fragmentation pattern. The degradation products formed under different stress conditions showed molecular masses of 443, 477, 433, 479, 463, and 459. The molecular mass of the detected impurity was 525. The drug and its related substances were efficiently separated on an Eclipse Plus C18 column (4.6 × 100 mm, 3.5 µm). An isocratic mobile phase consisting of acetonitrile and 0.01 M ammonium formate in water containing 0.1% formic acid (26:74, v/v) was used at a flow rate of 0.5 mL/min. Erdafitinib was exposed to oxidative, photolytic, acidic, basic, and thermal hydrolysis as recommended by ICH guidelines. The drug was found to be more sensitive to oxidative hydrolysis and slightly sensitive to acidic and photolytic hydrolysis. The method was fully validated according to ICH guidelines. The limit of detection and quantitation were 0.2 and 0.5 µg/mL, respectively. The relative standard deviations of intraday and interday precisions were not more than 1.99%. The developed method was successfully applied for the determination of erdafitinib in bulk and laboratory-prepared tablets.

NEW VALIDATED STABILITY-INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF METFORMIN HYDROCHLORIDE, LINAGLIPTIN AND EMPAGLIFLOZIN IN BULK AND PHARMACEUTICAL DOSAGE FORMS

Objective: The purpose of the present study is to develop simple, fast, accurate, precise, and robust stability-indicating reverse phase high-performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of metformin HCl, empagliflozin, and linagliptin in their combinations. Methods: Separation was performed on Agilent Eclipse XDB-C18 (250 mm x 4.6 mm, 5 µm) column with a mobile phase consisting of 0.1 % triethylamine (pH =3) buffer and acetonitrile in the ratio 40: 60 (v/v) at a flow rate of 1 ml/min. Detection of the analytes was carried out at a wavelength of 240 nm with a photodiode array detector. The developed method was validated as per the International Conference on Harmonization (ICH) guidelines. Results: The retention time values under the optimized condition were 2.660 min, 3.586 min, and 5.412 min for metformin HCl, linagliptin, and empagliflozin, respectively. The method was linear over a concentration range of 100 µg/ml-1500 µg/ml, 0.5 µg/ml-7.5 µg/ml, and 2.5 µg/ml-37.5 µg/ml for metformin HCl,linagliptin and empagliflozin respectively. The limit of detection (LOD) of the method was found to be 4.00 µg/ml, 0.02 µg/ml, and 1.00 µg/ml for metformin HCl, linagliptin, and empagliflozin, respectively. The degradation peaks were clearly resolved from the parent drug peaks in the chromatograms of forced degradation studies. Conclusion: The validated method was successfully applied for the determination of metformin HCl, linagliptin, and empagliflozin in their combined tablet dosage forms and hence can be used for the routine quality control of the drugs in pharmaceutical bulk, and dosage forms.

Development and Validation of Stability Indicating RP-HPLC Method for Simultaneous Determination of Doxycycline and Rifampicin in Polymeric Nanoparticles

Background: The combination of doxycycline (DOXY) and rifampicin (RIF) is recom-mended as a treatment therapy for brucellosis by the World Health Organization. Objective: The aim of the current study was to develop and validate the stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the analysis of a combina-tion of doxycycline &amp; rifampicin. Methods: The RP-HPLC method was developed and validated to estimate the doxycycline and ri-fampicin combination as per ICH guidelines. The drug combination solution was exposed to differ-ent stress conditions: acidic, basic, photo-oxidation, and oxidation. Results: The method was found linear in the range of 2 -10μg/mL for both the drugs with a reten-tion time of 3.5 min for doxycycline and 6.5 min for rifampicin at lambda maximum of 350 nm. The RP-HPLC method was precise and accurate with %RSD &lt; 2%. The intra-day and inter-day precisions were calculated and found within the acceptable range of 5%. Both drugs demonstrated good stability in the mobile phase after 6 hours. The LOD and LOQ of doxycycline and rifampicin were 100 ng/mL &amp; 200ng/mL and 150ng/mL &amp; 500ng/mL, respectively. The forced degradation of the combination of drug solutions was performed. The degraded drug peaks were well-resolved from the peaks of drugs. The percentage encapsulation efficiency of doxycycline and rifampicin in the nanoparticle system was assessed by utilizing the validated RP-HPLC method and found &gt;60% (DOXY) and &gt;70% (RIF). Conclusion: The developed RP-HPLC method of DOXY-RIF combination was rapid, accurate, precise, and stability-indicating. The method can be appropriately applied todetecte drugs in the na-noparticulate system.