Reverse-phase Ultra-performance Liquid Chromatographic Method Research Articles

Improved Stability-Indicating RP-UPLC Method for the Levamisole Hydrochloride Assay and Estimation of Its Related Compounds.

Levamisole hydrochloride (LVM) is an anthelmintic drug substance with immunomodulatory and anticancer activities. LVM has also found usage as a cutting agent in street cocaine. This study was aimed to develop and validate an alternative and improved stability-indicating reversed-phase ultraperformance liquid chromatography (RP-UPLC) method for the determination of LVM and the estimation of its related compounds. The UPLC method for the assay was optimized in terms of organic solvents consumed, pH, column temperature, and flow rate. Determination of LVM and its related compounds was performed using a gradient elution on a Waters ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm i.d., 130 Å). The column temperature was maintained at 35°C. Mobile phase A was composed of aqueous 5 mM ammonium hydroxide, and mobile phase B was composed of acetonitrile. All the analytes were monitored by UV detection at 215 nm with a flow rate of 0.7 mL/min. The total runtime of the method with column equilibration is 4.0 min. The developed method met all the acceptance criteria of the current International Council for Harmonization [ICH Q2 (R1)] guidelines. The method was tested in terms of specificity, linearity (R2 > 0.999), limit of detection (LOD; 0.06 μg/mL), limit of quantitation (LOQ; 0.2 μg/mL), accuracy, precision, and robustness. With a short analysis time (<2.5 min) and reduced consumption of organic solvents, the proposed method is considered a greener alternative to conventional chromatographic methods. An alternative and improved UPLC method was successfully developed and validated in accordance with the ICH guidelines for the determination of LVM and the estimation of its related compounds. Due to its high degree of selectivity, speed, and accuracy, the developed method can significantly benefit the end-users with laboratory efficiency and throughput during routine analysis of production batches and stability monitoring of LVM-related drug products.

A Chemometric Strategy in the Development of a RP-UPLC Method for the Quantitative Resolution of a Two-Component Syrup Formulation.

A novel chemometric strategy was implemented in the development of a new ultraperformance liquid chromatography method for the quantitative estimation of guaifenesin and pseudoephedrine hydrochloride in a two-component syrup formulation with minimal experimental effort, time and reagent. A full factorial design with three factors was investigated to find optimal working conditions of chromatographic factors (column temperature, flow rate, and 0.1M H3PO4% in mobile phase) that affect the chromatographic separation. Then, optimum experimental conditions providing adequate separation of the analyzed drug substances within the short runtime were determined. Under optimal experimental conditions, the retention times for guaifenesin and pseudoephedrine hydrochloride were obtained as 0.817 and 1.430min, respectively. In the optimized RP-UPLC method, chromatographic response was reported as a linear function of concentration between 5.0 and 80.0μg/mL for guaifenesin and 10.0-90.0μg/mL for pseudoephedrine hydrochloride. The proposed method was carefully validated and successfully applied to quality control and analysis of a cough syrup preparation containing guaifenesin and pseudoephedrine hydrochloride. Consequently, the proposed reversed-phase ultraperformance liquid chromatography method provided an opportunity to quantify relevant drugs with small amount of reagents and short runtime.

Stability-Indicating New RP-UPLC Method for Simultaneous Determination of a Quaternary Mixture of Paracetamol, Pseudoephedrine, Chlorpheniramine, and Sodium Benzoate in (Cold-Flu) Syrup Dosage Form.

A combination of paracetamol, pseudoephedrine, chlorpheniramine, and sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form is specified for the treatment of common cold and flu symptoms. The functional role of this study is to develop a novel, reliable, and selective stability-indicating reversed-phase ultra-performance liquid chromatography (RP-UPLC) method for simultaneous identification of a quaternary mixture of paracetamol, pseudoephedrine, chlorpheniramine, and sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form. The specific method is accomplished using an Acquity UPLC HSS T3 C18 column (2.1 mm × 100 mm), 1.8 µm particle size with pore size 100 Å, utilizing a mixture of purified water-methanol-trifluoroacetic acid (72.5:27.5:1.5, v/v) as the mobile phase at a flow rate of 0.3 mL/min. The column void volume is 1.15 min. UPLC detection is adjusted at 205 nm using a photodiode array detector. Calibration curves are obtained in the linearity ranges: 25-500 µg/mL for paracetamol, 10-50 µg/mL for pseudoephedrine, 0.5-5 µg/mL for chlorpheniramine, and 3-30 µg/mL for sodium benzoate with a correlation coefficient > 0.9992. The mean recovery of the developed method is tested and shows good recovery results between 99-101%; selectivity and forced degradation studies are investigated as per the International Council for Harmonisation Guidelines and no interference is detected due to degradation peaks. The proposed stability-indicating UPLC method for simultaneous determination of the three drugs, paracetamol, pseudoephedrine, and chlorpheniramine, with a preservative sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form is successfully accomplished, developed, and validated, and can be easily used in the analysis of drugs in pure or dosage form. The novelty of the current research work lies in the development of the UPLC method for simultaneous determination of a quaternary mixture of paracetamol, pseudoephedrine, chlorpheniramine, and sodium benzoate in (Cold-Flu) 1,2,3 Syrup dosage form.

A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles.

High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.

Development of new stability indicating UPLC-UV method for the extraction and quantification of perindopril and indapamide from human plasma

BackgroundThe hypertension and cardiovascular ailments are the leading cause of deaths worldwide. The combination therapy was found to be effective on the cardiovascular illness by reducing the blood pressure. The indapamide and perindopril combination therapy showed excellent results on reducing high blood pressure. With this in mind, the stability indicating reverse phase UPLC method was developed for the simultaneous identification and quantification of indapamide and perindopril from human plasma. In this work, we developed a new solid phase extraction method for the extraction of indapamide and perindopril in human plasma. It is a simple, accurate, and selective method for the extraction of these two drugs from human plasma with elution time of 2 min. The extracted drugs were identified and quantified by using stability indicating UPLC method. The method showed high recovery rate as well as low detection and quantification limits of two drugs.ResultsA novel, simple, highly accurate, and precise stability indicating ultra-performance liquid chromatography (UPLC) method was developed for the identification and quantification of perindopril (PP) (brand name Coversyl) and indapamide (IP) (brand name Lorvas) from human plasma. In this UPLC method, HSS C18 column (100 × 2.1 mm, 1.8 μm) and mobile phase acetonitrile (ACN), 10 mM KH2PO4 buffer solution (pH 3.0) mixture was used in the ratio of 65:35. Colum temperature of 30 °C, flow rate of 1.0 mL per minute and UV wave length of 254 nm were used. PP and IP were eluted below 2 min runtime with high resolution. Solid phase extraction (SPE) method was used for the extraction of PP and IP from human plasma. Different solvents were used to extract the analyte from SPE such as ACN, methanol, acetone, tertiary butyl diethyl ether (TBDE), chloroform (CHCl3), and ethanol (EtOH). Among these, ACN gave good recovery percentages (94.56 to 101.58%). From the linearity graph, good correlation coefficient values of 0.9996 for PP and 0.9997 for IP were achieved. The coefficient variance values for intra and inter day precision is in between 1.08 and 12.5%. The LOD and LOQ values were determined by the signal to noise ratio method. LOD and LOQ values for IP and PP were found to be 8.6 and 33.5 ng/mL and 28.33 and 110.5 ng/mL respectively. The developed method was statistically validated as per ICH guidelines.ConclusionIn summary, a novel stability indicating UPLC-UV method was developed and validated for the simultaneous identification and quantification of perindopril and indapamide drugs in human plasma and tested the stability as per ICH guidelines. It is a simple, accurate, and specific method for the extraction of these two drugs from human plasma and eluted within 2 min runtime. The method showed high recovery rate as well as low detection and quantification limits of two drugs. The developed method is suitable for routine analysis as well as in bioanalytical and clinical studies.Graphical abstract

Elucidation of plasma protein binding, blood partitioning, permeability, CYP phenotyping and CYP inhibition studies of Withanone using validated UPLC method: An active constituent of neuroprotective herb Ashwagandha

Ethnopharmacological relevanceWithanone (WN), an active constituent of Withania somnifera commonly called Ashwagandha has remarkable pharmacological responses along with neurological activities. However, for a better understanding of the pharmacokinetic and pharmacodynamic behavior of WN, a comprehensive in-vitro ADME (absorption, distribution, metabolism, and excretion) studies are necessary. Aim of the studyA precise, accurate, and sensitive reverse-phase ultra-performance liquid chromatographic method of WN was developed and validated in rat plasma for the first time. The developed method was successfully applied to the in-vitro ADME investigation of WN. Material and methodsThe passive permeability of WN was assayed using PAMPA plates and the plasma protein binding (PPB) was performed using the equilibrium dialysis method. Pooled liver microsomes of rat (RLM) and human (HLM) were used for the microsomal stability, CYP phenotyping, and inhibition studies. CYP phenotyping was evaluated using the specific inhibitors. CYP inhibition study was performed using specific probe substrates along with WN or specific inhibitors. ResultsWN was found to be stable in the simulated gastric and intestinal environment and has a high passive permeability at pH 4.0 and 7.0 in PAMPA assay. The PPB of WN at 5 and 20 μg/mL concentrations were found to be high i.e. 82.01 ± 1.44 and 88.02 ± 1.15%, respectively. The in vitro half-life of WN in RLM and HLM was found to be 59.63 ± 2.50 and 68.42 ± 2.19 min, respectively. CYP phenotyping results showed that WN was extensively metabolized by CYP 3A4 and1A2 enzymes in RLM and HLM. However, the results of CYP Inhibition studies showed that none of the CYP isoenzymes were potentially inhibited by WN in RLM and HLM. ConclusionThe in vitro results of pH-dependent stability, plasma stability, permeability, PPB, blood partitioning, microsomal stability, CYP phenotyping, and CYP inhibition studies demonstrated that WN could be a better phytochemical for neurological disorders.

The QUALITY BY DESIGN APPROACH FOR SIMULTANEOUS DETERMINATION OF FLUTICASONE PROPIONATE AND SALMETEROL XINAFOATE

Objective: To develop and validate a novel and simple reverse phase Ultra Performance Liquid Chromatography (UPLC) method for simultaneous determination of Fluticasone Propionate and Salmeterol Xinafoate from pharmaceutical finished product, applying Quality by design (QbD) approach.&#x0D; Methods: The proposed analytical method developed and validated in a linear gradient condition at a flow rate of 0.40 ml/min over Waters ACQUITY BEH Shield RP 18, 2.1*100 mm, 1.7 µm column by maintaining column oven temperature at 30 °C and Sample cooler temperature at 15 °C. Chromatograms monitored and recorded at 215 nm.&#x0D; Results: The proposed method has been validated as per International Conference on Harmonization (ICH) guidelines with respect to system suitability, specificity, precision, linearity, accuracy, range, solution stability and robustness. This method is qualified in all parameters in case of system suitability and specificity; precision observed within the limit of 2.0%, the excellent linear response observed with correlation coefficient (R2) for Salmeterol 0.99999 and Fluticasone Propionate 0.99999, for Accuracy within the limit of 98% to 102%.&#x0D; Conclusion: A selective, suitable and accurate reverse phase UPLC method for simultaneous Determination of Fluticasone Propionate and Salmeterol Xinafoate in the pharmaceutical finished product has been developed and validated successfully.

Development and Validation of Novel and Highly Sensitive Stability-Indicating Reverse Phase Ultra Performance Liquid Chromatography Method for Quantification of Ibrutinib and its ten Degradation Products

In present study, a novel, simple, robust, stability-indicating, highly sensitive and specific reverse phase ultra-performance liquid chromatographic method was established and validated for the determination of ibrutinib using acquity charged surface hybrid C-18 (100 mm×2.1 mm, 1.7 μm) column with a mobile phase consisting of mixture of Eluent-A phosphate buffer, 0.1 % triethylamine, (pH 6.0) and Eluent-B acetonitrile with flow rate 0.3 ml min-1. The detection was monitored at 215 nm using photodiode array detector. Linearity was observed from 25 to 250 ng ml-1 (correlation of coefficient was 0.9999) with equation, y=100.1880x-130.8966. The observed limit of detection and limit of quantitation were 25 ng ml-1 and 50 ng ml-1 respectively. Ibrutinib was subjected to stress conditions comprising hydrolytic (acid, alkali and neutral), oxidative, photolytic and thermal degradation as per International council for harmonisation Q1 guidelines. The results showed that substantial degradation were observed in acid, base and peroxide degradation condition and resistant to neutral, photolytic and thermal degradation. This is first highly sensitive stability-indicating ultra-performance liquid chromatography method capable of separating ibrutinib and its ten degradation products at nano gram (ng) level. The method was validated as stated by International council for harmonisation guidelines.

Development of stability indicating method for the simultaneous estimation of alogliptin and pioglitazone in bulk and tablet dosage form by reversed-phase ultra-performance liquid chromatography method

Development of stability indicating method for the simultaneous estimation of alogliptin and pioglitazone in bulk and tablet dosage form by reversed-phase ultra-performance liquid chromatography method

A Validated Ultra-Performance Liquid Chromatographic Method for the Simultaneous Determination of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate in Their Combined Dosage Form and Spiked Human Plasma Samples.

Nadifloxacin, mometasone furoate and miconazole nitrate are formulated together as a topical antifungal dosage form. In this work, a reversed-phase ultra-performance liquid chromatographic method coupled with a diode array detector (RP-UPLC-DAD) was developed and validated to determine nadifloxacin, mometasone furoate and miconazole nitrate simultaneously in their bulk powder, in pharmaceutical preparation and in spiked human plasma samples. Separation was achieved on an ACQUITY UPLC C18 column of 2.2μm particle size (2.1×100mm) via isocratic elution using a mobile phase consisting of methanol, acetonitrile and water with ratio (50:20:30; v/v/v) and 0.1g ammonium acetate, then pH was adjusted to (7.00) using acetic acid, flow rate 0.6mL/min, temperature 30°C and UV detection at 220nm. The method is linear in a range from 5 to 400μg/mL for both nadifloxacin and miconazole nitrate and from 20 to 500μg/mL for mometasone furoate. The method was validated according to the ICH guidelines then applied successfully to determine the mentioned drugs in their pharmaceutical preparation and spiked human plasma samples. For plasma samples, the results showed that the method can determine nadifloxacin, mometasone furoate and miconazole nitrate in human plasma samples with high accuracy and precision.

Analysis of polyhexamethylene biguanide and alexidine in contact lens solutions using capillary electrophoresis, ultra-performance liquid chromatography and quadrupole time of flight mass spectrometry

Polymeric biguanides, as well as quaternary ammonium compounds, are ubiquitous antimicrobial agents in healthcare. Due to the highly cationic and polymeric nature of these compounds and the complex matrices in which they are found, the analytical characterization of products containing them remains challenging. In this work an efficient, sensitive, and high-resolution separation protocol was developed to perform quantitative measurements (sub-mg L−1) of alexidine dihydrochloride (ADH) and polyhexamethylene biguanide (PHMB) in commercial multipurpose contact lens solutions (MPS). Initially, contactless conductivity (C4D) detection was explored, but lacked adequate selectivity and sensitivity to quantify PHMB or ADH in commercial MPS. To overcome these limitations, an alternative approach using solid phase extraction (SPE) followed by separation with reversed phase ultra-performance liquid chromatography (RP-UPLC) was developed for both ADH and PHMB separation and detection. The most sensitive and reliable method investigated utilized standard additions to compensate for matrix effects. For ADH, concentration values measured with the presented method were consistent with data provided by the MPS manufacturer (1.6 mg L−1) within 0.10 mg L−1. PHMB quantification in MPS products was successful at concentrations <1 mg L−1 with quantitative reproducibility better than 2% RSD. Comparison of blind sample testing using the RP-UPLC method showed strong correlation (R2 = 0.939) of PHMB concentrations with results obtained by the United States Food and Drug Administration using a published HPLC-Evaporative light scattering detection (ELSD) assay. A significant advantage of this method is the ability to partially resolve PHMB polydispersity, which to date has been minimally studied and explained. By coupling with electrospray mass spectrometry (MS), a general trend was observed for increased retention as a function of PHMB chain length. The improved robustness and reproducibility of UV detection versus ELSD coupled with the superior resolving power of UPLC is an asset to the detection and characterization of PHMB and ADH. In addition to quality control of MPS, this method has potential application to the analyses skin wipes, wound dressings and other medical products where understanding how manufacturing processes lead to differences in polydispersity is important to maximize the antimicrobial properties while minimizing toxicologic effects.

Improved IC-50 prediction using the Quasi Vivo® in vitro dynamic cell culture flow system

Withanone (WN), an active constituent of Withania somnifera commonly called Ashwagandha has remarkable pharmacological responses along with neurological activities. However, for a better understanding of the pharmacokinetic and pharmacodynamic behavior of WN, a comprehensive in-vitro ADME (absorption, distribution, metabolism, and excretion) studies are necessary.A precise, accurate, and sensitive reverse-phase ultra-performance liquid chromatographic method of WN was developed and validated in rat plasma for the first time. The developed method was successfully applied to the in-vitro ADME investigation of WN.The passive permeability of WN was assayed using PAMPA plates and the plasma protein binding (PPB) was performed using the equilibrium dialysis method. Pooled liver microsomes of rat (RLM) and human (HLM) were used for the microsomal stability, CYP phenotyping, and inhibition studies. CYP phenotyping was evaluated using the specific inhibitors. CYP inhibition study was performed using specific probe substrates along with WN or specific inhibitors.WN was found to be stable in the simulated gastric and intestinal environment and has a high passive permeability at pH 4.0 and 7.0 in PAMPA assay. The PPB of WN at 5 and 20 μg/mL concentrations were found to be high i.e. 82.01 ± 1.44 and 88.02 ± 1.15%, respectively. The in vitro half-life of WN in RLM and HLM was found to be 59.63 ± 2.50 and 68.42 ± 2.19 min, respectively. CYP phenotyping results showed that WN was extensively metabolized by CYP 3A4 and1A2 enzymes in RLM and HLM. However, the results of CYP Inhibition studies showed that none of the CYP isoenzymes were potentially inhibited by WN in RLM and HLM.The in vitro results of pH-dependent stability, plasma stability, permeability, PPB, blood partitioning, microsomal stability, CYP phenotyping, and CYP inhibition studies demonstrated that WN could be a better phytochemical for neurological disorders.

Simultaneous determination of ascorbic acid and caffeine in commercial soft drinks using reversed-phase ultraperformance liquid chromatography

A new reversed-phase ultraperformance liquid chromatography method with a photodiode array detector was developed for the quantification of ascorbic acid (AA) and caffeine (CAF) in 11 different commercial drinks consisting of one energy drink and 10 ice tea drinks. Separation of the analyzed AA and CAF with an internal standard, caffeic acid, was performed on a Waters BEH C18 column (100 mm × 2.1 mm, 1.7 μm i.d.), using a mobile phase consisting of acetonitrile and 0.2M H3PO4 (11:89, v/v) with a flow rate of 0.25 mL/min and an injection volume of 1.0 μL. Calibration graphs for AA and CAF were computed from the peak area ratio of AA/internal standard and CAF/internal standard detected at 244.0 nm and 273.6 nm, respectively. The developed reversed-phase ultraperformance liquid chromatography method was validated by analyzing standard addition samples. The proposed reversed-phase ultraperformance liquid chromatography method gave us successful results for the quantitative analysis of commercial drinks containing AA and CAF substances.

Development and Validation of RP-UPLC Method for the Determination of Process and Degradant Impurities Present in Dabigatran Etexilate Mesylate Capsules Using High Strength Silica-T3 Sorbent Column

A Reverse phase-Ultra Performance liquid chromatography (RP-UPLC) method was developed and validated for the quantification of process and degradant impurities present in Dabigatran etexilate mesylate capsules. Degradation data in each condition shows that peaks are pure and method is found to be stability indicating in nature. Impurities are well separated at satisfactory level in Acquity High strength silica (HSS)-T3, 100 mm length x 2.1 mm ID, Octadecyl column with 1.8μ particle size. Mobile phase-A consists of 0.1% Orthophosphoric acid in water adjusted the pH to 3.5 with triethyl amine. Acetonitrile used as Mobile phase-B. Gradient elution mode has been chosen to elute all impurities of Dabigatran etexilate which are having different polarities. Finalized flow rate was 0.18 mL.min‒1 with a quantification wavelength of 290 nm. The developed method was successfully validated for specificity, linearity, precision, accuracy, determination of LOD, LOQ and robustness as per International conference on harmonization (ICH) general guidelines requirement. The validated method is useful for quantitative determination of impurities of Dabigatran etexilate in Dabigatran etexilate capsules dosage form, which enables to identify the therapeutic efficacy at reduced run time.

Validated Stability-indicating Reverse-phase Ultra-performance Liquid Chromatography Method for Simultaneous Determination of Sodium Methylparaben, Sodium Propylparaben and Ketorolac Tromethamine in Topical Dosage Forms.

A sensitive, fast, and stability-indicating isocratic reverse-phase ultra-performance liquid chromatography method was developed and validated for quantitative simultaneous determination of sodium methylparaben, sodium propylparaben and ketorolac tromethamine in topical dosage forms. Separation of all peaks was achieved by using acquity ethylene bridged hybrid C18 (50×2.1 mm, 1.7 μ) as stationary phase, mobile phase used was triethylamine buffer (pH 2.5):tetrahydrofuran:methanol (665:35:300, v/v/v) with isocratic mode at a flow rate of 0.40 ml/min. All component were detected at 252 nm with 10 min run time. The described method was found to be linear in the concentration range of 248-744 μg/ml for ketorolac tromethamine, 20.8-62.4 μg/ml for sodium methylparaben and 2.38-7.13 μg/ml for sodium propylparaben with correlation coefficients more than 0.999. Method was validated in terms of specificity, linearity, accuracy, precision, solution stability, filter equivalency, and robustness as per International Conference on Harmonization guideline. Formulation was exposed to the stress conditions of peroxide, acid, base, thermal, and photolytic degradation and proven all components were well separated in the presence of degradants.

LC–MS/MS characterization of the forced degradation products of ezetemibe: Development and validation of a stability-indicating UPLC method

The present study reports the characterization of four degradation products from ezetemibe (EZB) through liquid chromatography–tandem mass spectrometry (LC–MS/MS) and the development of a validated and stability-indicating reversed-phase ultra-performance liquid chromatographic method for the determination of EZB in the presence of its process-related impurities in bulk drugs. The forced decomposition of EZB was carried out and studied under acidic, basic, oxidative, photolytic and thermal conditions. The degradation of EZB was observed under basic and acidic conditions, and four degradation products (DPs) were formed. Successful chromatographic separation of EZB and its degradation products were successfully separated through chromatography with a Waters Acquity HSST3 C18 stationary phase (50×2.1mm, 1.7μm). The analytes were detected with a PDA detector set at 230nm. The figures of merit for this method were adequate. The assay remained linear from concentrations of 0.09μgmL−1 to 600μgmL−1 for EZB and its four DPs (r2=0.99914, 0.99945, 0.99917, 0.99923 and 0.99936 for EZB, Imp-A, Imp-B, Imp-C, and Imp-D, respectively). The method precision, which was expressed as the %RSD, ranged from 0.2 to 1.0 for the four impurities. The DPs from EZB were characterized by LC–MS/MS, and the most likely degradation and fragmentation pathways for EZB and its DPs were proposed.

Determination of lupenone and β-sitosterol in Rhizoma Musae by UPLC with DAD and ELSD.

Rhizoma Musae has been used for centuries in Miao medicine practice in China, and it usually uses for treating diabetes and bruises. In this study, lupenone and β-sitosterol in Rhizoma Musae were separated by reversed-phase ultra-performance liquid chromatography (RP-UPLC) and simultaneously detected by a diode array detector (DAD) and an evaporative light scattering detector (ELSD) using methanol and 0.1% aqueous acetic acid (100 : 4, v/v) as a mobile phase in 20 min. The flow rate of 0.1 mL/min was set with isocratic, the temperature of column compartment maintained at 50°C and ultraviolet detection set at 206 nm wavelength. The injection volume was 1.0 µL. The parameter for the ELSD was set to a probe temperature of 45°C, and the nebulizer for nitrogen gas was adjusted to 1.5 L/min. The RP-UPLC method was validated for accuracy, precision, limit of detection and limit of quantification. It applied to the quantification of the active chemical constituents of Rhizoma Musae, and results indicated that both DAD and ELSD were suitable for the determination of lupenone and β-sitosterol, and the DAD has a better sensitivity than the ELSD.

A New RP-UPLC Method for Simultaneous Quantitative Estimation of Bisoprolol Hemifumarate and Hydrochlorothiazide in Tablets Using Experimental Design and Optimization

A new reverse phase ultra-performance liquid chromatography (RP-UPLC) method was developed for the quantification of bisoprolol hemifumarate (BIS) and hydrochlorothiazide (HCT) in tablets using experimental design and optimization. In the development of method, a 33 full factorial design was used to identify the effects of factors (column temperature, flow rate, and 0.1 M H3PO4 % in mobile phase) and their interactions on the chromatographic response and to determine optimal chromatographic conditions providing a good peak elution and shortest runtime, respectively. For the optimal RP-UPLC conditions, 58.2°C (column temperature), 0.37 mL/min (flow rate) and 23.6% (0.1 M H3PO4) were obtained from the results of 33 full factorial design. The separation of BIS (retention time: 0.323 min) and HCT (retention time: 0.409 min) was performed within a short runtime of 0.6 min. Calibration curves for BIS and HCT in the range 8.0–40.0 µg/mL and 2.0–26.0 µg/mL were computed by the detection at 225 nm. The validity of the method was achieved by analyzing the test samples, and using inter-days and intra-day studies. Under optimal conditions, new RP-UPLC method was successfully applied for the analysis of BIS and HCT in commercial tablets.

Development and Validation of a Stability-Indicating RP-UPLC Method for the Estimation of Impurities in Cinacalcet Hydrochloride API and its Formulation.

A sensitive, stability-indicating, gradient reversed-phase ultra-performance liquid chromatography method has been developed for the quantitative estimation of cinacalcet hydrochloride impurities in active pharmaceutical ingredients and pharmaceutical formulations. Efficient chromatographic separation was achieved on an Acquity BEH Shield RP18, 100 × 2.1 mm, 1.7 µm column with the mobile phase containing pH 6.6 phosphate buffer and acetonitrile. The flow rate of the mobile phase was 0.3 mL min−1 with a column temperature of 35°C and detection wavelength at 223 nm. The relative response factor values of (+)-R-1-(1-Naphthyl)ethylamine, regioisomer, diastereomer isomer-1, and diastereomer isomer-2 were 1.79, 0.99, 0.89, and 0.88, respectively. The cinacalcet hydrochloride formulation sample was subjected to the stress conditions of acid, base, oxidative, hydrolytic, thermal, humidity, and photolytic degradation. Cinacalcet hydrochloride was found to degrade significantly under the peroxide stress conditions. The degradation products were well-resolved from cinacalcet hydrochloride and its impurities. The peak purity test results confirmed that the cinacalcet hydrochloride peak was homogenous in all stress samples and the mass balance was found to be more than 96%, thus proving the stability-indicating power of the method. The developed method was validated according to ICH guidelines.

Stability Indicating RP-UPLC Method for Assay of Emtricitabine and Tenofovir Disoproxil Fumarate in Bulk and Dosage Forms

A simple, sensitive and rapid stability indicating reverse phase ultra performance liquid chromatography (RP-UPLC) method was developed and validated for the determination of Emtricitabine (EMT) and Tenofovir disoproxil fumarate (TDF) in pure and tablet dosage forms. The chromatographic separation was achieved by using Waters (Alliance) UPLC system equipped with auto-sampler and photo diode array detector. A volume of 5 μL of standard or test was injected into the column and the components were separated by using the mixture of 0.68% potassium dihydrogen orthophosphate buffer of pH = 6 and methanol in the ratio 45:55 v/v as mobile phase at a flow rate of 1.2 mL/min through BEH C18 (100 mm × 2.1, 1.8 μm) at ambient temperature and were detected at a wavelength of 261 nm. System suitable parameters such as plate count and tailing factor for EMT and TDF were found to be 2427 & 3685, 1.16 & 1.23 respectively, and resolution between EMT and TDF peaks was found to be 3.12. The chromatographic parameters like retention time, peak area and peak height of EMT and TDF were found to be 0.684 & 0.930, 694,200 & 8,778,000 and 272,881 & 3685 respectively. Percent of assay of EMT and TDF in bulk and dosage forms was determined and found to be 101.48 and 103.22 respectively. Study of degradation was examined and found that the drugs were stable under degradation conditions. The present method was developed keeping the principles of green chemistry by using eco-friendly solvent methanol in mobile phase. The developed method was found to be simple, rapid and applied for the analysis of Truvada; therefore the proposed method is recommended for the analysis of EMT and TDF in pure and tablet dosage forms in any quality control laboratories.