XTerra C18 Research Articles

Identification and Quantification of Related Impurities of a Novel Ketolide Antibiotic Nafithromycin

Nafithromycin (NFT, WCK 4873) is a novel antimicrobial drug being developed to treat infections caused by Gram-positive bacteria. It is a second-generation drug of the lactone ketolide class. A mass spectrometry-compatible reversed-phase liquid chromatographic method was developed to separate and identify the impurities in NFT. Forced degradation (FD) study ensured specificity of the method and revealed the probable degradation products. Seven process impurities and five degradation products were chromatographically resolved and identified based on the protonated molecule peak and product ions. Process impurities were synthesized and characterized for identity and purity. Separation was achieved on the XTerra C18 stationary phase using the binary gradient method. Mobile phase ‘A’ constituted of a mixture of an aqueous solution of ammonium acetate (pH 9.0), acetonitrile and methanol in the ratio of (48:27:25, v/v/v). Mobile phase ‘B’ constituted of an aqueous solution of ammonium acetate (pH 9.0) and acetonitrile in the ratio of (05:95, v/v). The method was validated for accurate and precise quantification of the impurities. The developed method was employed for quality control and stability studies of the NFT drug substance used for pre-clinical and clinical studies.

RP-HPLC METHOD VALIDATION AND DEVELOPMENT FOR THE ESTIMATION OF PRULIFLOXACIN IN PHARMACEUTICAL DOSAGE FORM

RP-HPLC METHOD VALIDATION AND DEVELOPMENT FOR THE ESTIMATION OF PRULIFLOXACIN IN PHARMACEUTICAL DOSAGE FORM Mishra Shambhu Nath*, Dr. Omray Lavakesh, Mr. Soni Pushpendra Radharaman Institute of Pharmaceutical Sciences, Bhopal, Madhya Pradesh, India ABSTRACT The current research study, a successful practice was done for “Validated HPLC method development for the estimation of Prulifloxacin in marketed formulation”. Analytical method validation of Prulifloxacin tablets was developed via material and method plan which is based on logical experimental procedure through literature survey associated with statistical tools. Major instrument which was used is UV spectrophotometer (make shimadzu) and HPLC system was Shimadzu LC-2010CHT with Chromalion software. The isocratic mobile phase consisted of mixture of phosphate buffer preparation and acetonitrile in the ratio of 150:850 ml v/v and with PH 7.4 by adding triethylamine. Chromatographic parameters was follow is Column Xterra C18 125Å (250mm x 4.6mm x 0.5µm), Flow rate 1 ml/min, Injection volume 20µl, Column oven temperature 35oC, Sampler temperature 2-8oC and Run time 10 minute. Assay of prulifloxacin tablets (Alpruli 600 mg) in three set occur average %age is 100.63, all value between 98-102%. Proposed method was found to be linear in the range of 20-100 μg/ml prulifloxacin with the correlation coefficient near to one respectively. The validation and the reliability of proposed method was assessed by recovery study. The recovery of added standards (80%, 100% 120%) 99.7 %, 100.7 % and 100.4 % for prulifloxacin respectively. found prulifloxacin is stable in low temperature between 2-8oC upto 48 Hours. Keywords: Ulifloxacin, RP-HPLC, Stress testing. Antimicrobial, Non-polar Full Text Article

Validation of developed method by RP-HPLC for estimation of Prasugrelin human plasma and studying the stability of the drugs in plasma

This paper is concern with a reverse phase high performance liquid chromatography (RP-HPLC) bio-analytical method development and validation for Prasugrel in human plasma using photo diode array detector (PDA detector). The HPLC separation was carried out in an isocratic mode on an X-Terra C18 column (4.6 x 150 mm; 5 μm) with a mobile phase consisting of potassium dihydrogen phosphate [pH 3.0] and acetonitrile in the ratio of 30:70 v/v at a flow rate of 1.0 mL/min. The run time was maintained for 5 mins and the detection was monitored at 210 nm. The percentage recovery was found 99.61-100.06 in human plasma. This reveals that the method is quite accurate. The linearity was found 15-40 μg/mL in human plasma. The inter-day and intra-day precision in plasma was found within the limits. The lower limit of quantification (LLOQ) obtained by the proposed method was 0.05 μg/mL. The percentage relative standard deviation (%RSD) obtained for the drug spiked in plasma for stability studies were less than 2 %.Kathmandu University Journal of Science, Engineering and TechnologyVol. 13, No. 1, 2017, Page: 65-75

Simultaneous determination of dihydrotestosterone and its metabolites in mouse sera by LC-MS/MS with chemical derivatization

Androgens play a vital role in prostate cancer development, and their elimination and blockade are essential in the disease management. DHT is the key ligand for androgen receptor (AR) in the prostate. It is locally synthesized from testosterone. In the prostate, DHT is predominantly metabolized to α-diol and β-diol. Recent studies indicate that impaired DHT catabolism is associated with prostate cancer, signifying the necessity of a sensitive quantitative method for the determination of DHT and its metabolites.In this work, an LC-MS/MS method for the simultaneous quantification of DHT and its metabolites was developed and validated. Steroid-free sera were prepared and used for the preparation of sera calibrators and quality controls (QCs). DHT and its metabolites along with their respective stable heavy isotope labeled analytes representing internal standards were first extracted with methyl tertiary-butyl ether (MTBE) and derivatized with picolinic acid (PA). The derivatized analytes were then extracted again with MTBE, dried under nitrogen and reconstituted in the mobile phase (80% methanol and 0.2% formic acid in water). Baseline chromatographic separation of the derivatized analytes was achieved isocratically on XTerra C18 column (2.1 × 100 mm) using the mobile phase at a flow rate of 0.25 mL/min. Quantitation was performed using multiple-reaction-monitoring mode with positive electrospray ionization. The method has calibration ranges from 0.0500 ng/mL to 50.0 ng/mL for DHT and its two metabolites with acceptable assay precision, accuracy, recovery, and matrix factor. It was applied to the determination of DHT and its metabolites in an animal study.

Development and validation of LC-MS/MS assay for the determination of Butoconazole in human plasma: Evaluation of systemic absorption following topical application in healthy volunteers

Butoconazole is an imidazole antifungal that is more effective than miconazole and clotrimazole for treatment of vaginal candidiasis. A highly sensitive tandem mass spectrometric assay was developed and validated to evaluate systemic absorption of Butoconazole following intravaginal administration. Chromatographic separation was achieved using Waters Xterra C18 column (3µm, 3.0×50.0mm). Liquid-liquid extraction using tert-butyl methyl ether was used for preparation of plasma samples. The mobile phase was solvent A: 0.1% formic acid in water and solvent B: acetonitrile: methanol (30:70, v/v), using gradient elution mode at 0.5mL/min. Detection at positive electrospray ionization in the MRM mode was then employed. Analysis was carried out within 5.5min over a linear concentration range of 0.10–30.00ng/mL. Validation was carried out according to US FDA guidelines for bioanalytical method validation. Matrix effect, recovery efficiency and process efficiency have been investigated for the analyte and internal standard in neat solvent, post-extraction matrix and plasma. The mean percentage recoveries were higher than 80%, the accuracy was 93.51–106.85% and the RSD was below 10% throughout the studied concentration range. Results indicated sufficient stability of the target analyte in plasma at the employed experimental conditions. Results of incurred sample re-analysis and incurred sample stability revealed less than 5% variability. The applicability of the assay for monitoring of the systemic absorption of Butoconazole following intra vaginal application to healthy volunteers was demonstrated. Results confirmed that Butoconazole was detected shortly after intra vaginal administration with Cmax and tmax of 30ng/mL and 6h, respectively.

Method development and Validation of RP-HPLC method for the Determination of Olanzapine in Bulk and Tablet Dosage form

Objective: To develop and validate reverse phase-high performance liquid chromatographic method for estimation of olanzapine in bulk and tablet dosage form.Methods: Chromatographic analysis was performed on XTerra C18 (150×3.5 mm inner diameter, 5 μm) column using a mobile phase consisting of buffer (potassium dihydrogen phosphate) and methanol (45:55% v/v) with a flow rate of 0.6 ml/minutes. The detection was carried out at 247 nm.Results: The calibration curve of olanzapine was linear in the range of 30-70 μg/ml. The mean % assay of marketed formulation was found to be 100.2%, and % recovery was observed in the range of 98-102%. Relative standard deviation for the precision study was found <2%.Conclusion: The developed method is simple, precise and rapid, making it suitable for estimation of olanzapine in bulk and tablet dosage form.Keywords: Olanzapine, Reverse phase-high performance liquid chromatographic, Validation.

Bioanalytical Method Validation for Dronedarone and Duloxetine in Blood Serum

The present work relates to the development and validation of reversed-phase HPLC-UV-photodiode array methods for the estimation of two drugs in blood serum: dronedarone hydrochloride (DDN), a class III antiarrhythmic drug, and duloxetine hydrochloride (DLX), an antidepressant. Chromatographic analysis of DLX was carried out on a Nucleodur C18 column (250 × 4.6 mm, 5 μm) using ammonium acetate buffer (32 mM, pH 5.5) and acetonitrile (40 + 60, v/v; flow rate of 1.0 mL/min; detection wavelength of 290 nm) as the mobile phase. A Waters XTerra C18 column (250 × 4.6 mm, 5 μm) was used for the chromatographic analysis of DDN using an acetonitrile-ammonium formate buffer (20 mM, pH 3.0, with formic acid; 45 + 55, v/v; flow rate 1.0 mL/min) as the mobile phase. Pentazocine and bupropion HCl were used as the internal reference standards for DLX and DDN, respectively. Excellent linearity was observed for DLX (r2 = 0.9996; concentration range 0.2-10.0 μg/mL) and DDN (r2 = 0.9997; concn. range 2.0-50.0 μg/mL). The LODs for DLX and DDN were 0.022 and 0.78 μg/mL, respectively, and the LOQs 0.066 and 2.4 μg/mL, respectively.

Sensitive, automatic method for the determination of diazepam and its five metabolites in human oral fluid by online solid-phase extraction and liquid chromatography with tandem mass spectrometry.

A novel and simple online solid-phase extraction liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of diazepam and its five metabolites including nordazepam, oxazepam, temazepam, oxazepam glucuronide, and temazepam glucuronide in human oral fluid. Human oral fluid was obtained using the Salivette(®) collection device, and 100 μL of oral fluid samples were loaded onto HySphere Resin GP cartridge for extraction. Analytes were separated on a Waters Xterra C18 column and quantified by liquid chromatography with tandem mass spectrometry using the multiple reaction monitoring mode. The whole procedure was automatic, and the total run time was 21 min. The limit of detection was in the range of 0.05-0.1 ng/mL for all analytes. The linearity ranged from 0.25 to 250 ng/mL for oxazepam, and 0.1 to 100 ng/mL for the other five analytes. Intraday and interday precision for all analytes was 0.6-12.8 and 1.0-9.2%, respectively. Accuracy ranged from 95.6 to 114.7%. Method recoveries were in the range of 65.1-80.8%. This method was fully automated, simple, and sensitive. Authentic oral fluid samples collected from two volunteers after consuming a single oral dose of 10 mg diazepam were analyzed to demonstrate the applicability of this method.

Determination of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in poultry, swine, bovine and fish by liquid chromatography-tandem mass spectrometry

A simple, rapid and sensitive method for confirmatory and quantitative purposes using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) was developed and validated for determination of chloramphenicol, thiamphenicol, florfenicol and its metabolite, florfenicol amine, in poultry, swine, bovine and fish muscle. Sample preparation was based on extraction with organic solvent (ethyl acetate: ammonium hydroxide, 98:2) followed by evaporation and fat removal using hexane. The chromatographic separation was carried out with an XTerra C18 column with a gradient elution using water and acetonitrile both with 2mM of ammonium acetate. Mass spectrometry with electrospray ionization was operated in positive or negative polarity using selected-reaction monitoring (SRM) analysis mode, achieving the requirements of four identification points for each compound. Chloramphenicol-D5 was added as internal standard. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Parameters as precision, reproducibility, trueness, CCα and CCβ were determined. Trueness values were within the range 82–108% and 84–111% for bovine and fish, respectively. Precision ranged from 1.1% to 10.1% and within-laboratory reproducibility ranged from 4.3 to 18.1%, depending on matrix. The CCα and CCβ for bovine muscle, for instance, were established as 0.06 and 0.11μgkg−1, respectively.The method was successfully applied for several interlaboratory proficiency testing programs, achieving 100% of satisfactory results.

ESTIMATION OF ACECLOFENAC IN ALBINO RAT PLASMA BY USING RP-HPLC

A new RP-HPLC method for the quantitative determination of aceclofenac in albino rat plasma was developed and validated as per US-FDA guidelines. The drug was spiked in the plasma and extracted with mobile phase by precipitation method. The extracted analyte was injected into XTerra c18 (4.6 x 250 mm; 5 µm) or equivalent, maintained at 25°C temperature and effluent was monitored at 274 nm. The mobile phase consisted of potassium dihydrogen phosphate [ph 4.5]: methanol: acetonitrile [HPLC grade] (55:25:20 (V/V). The flow rate was maintained at 1.0 mL/min. The developed method showed high specificity for aceclofenac. The calibration curve for aceclofenac was linear from 25.0 to 125.0 ng/0.5mL plasma(r2= 0.999). The inter-day and intra-day precision was found to be within limits. The average % recovery for aceclofenac was found to be 99.74-99.77 % and the reproducibility was found to be satisfactory.The proposed method was adequate, sensitivity, reproducibility, and specificity for the determination of aceclofenac in albino rat plasma.

Development and Validation of a Sensitive RP-HPLC method for Simultaneous Estimation of Rosuvastatin and Fenofibrate in Tablet Dosage form by using PDA Detector in Gradient Mode

Objective: The present work was undertaken with the aim to develop and validate a rapid and consistent RP-HPLC method in which the peaks will be appear with short period of time as per ICH Guidelines. The proposed method was simple, fast, accurate and precise method for the Quantification of drug in the dosage form, bulk drug as well as for routine analysis in Quality control. Method: RP-HPLC method was developed and validated for simultaneous estimation of Rosuvastatin and Fenofibrate in bulk drug and in combined dosage forms. The HPLC separation was achieved on a XTerra C18 (4.6 X 150mm, 5?m, Make: Thermosil) or equivalent in an Isocratic Mode. The mobile phase was composed of Phosphate Buffer (25%) whose pH was adjusted to 3.0 by using Ortho Phosphoric Acid and Methanol (75%) [HPLC Grade] Results: The flow rate was monitored at 1.0ml per min. The wavelength was selected for the detection was 254 nm. The run time was 7min. The retention time found for the drugs Rosuvastatin and Fenofibrate were 1.997 min., 3.238 min. and 4.042 min. respectively. The % recovery was found to be 98.46% - 101.79% for the drug Rosuvastatin. The % recovery was found to be 98.04% - 101.79% for the drug Fenofibrate. The linearity was established in the range of 80 to 120ppm for the drug Rosuvastatin and 2.4 to3.6ppm for the drug Fenofibrate. The LOD for the drugs Rosuvastatin and Fenofibrate were found to be 0.07µg/ml, 0.07µg/ml and 0.006µg/ml respectively. The LOQ for the drugs Rosuvastatin and Fenofibrate were found to be 0.23µg/ml, 0.24µg/ml and 0.02µg/ml respectively. Conclusion: The proposed method was adequate sensitive, reproducible, and specific for the determination of Rosuvastatin and Fenofibrate in bulk as well as in Tablet dosage form. The validation of method was carried out utilizing ICH-guidelines. The described RP-HPLC method was successfully employed for the analysis of pharmaceutical formulations containing combined dosage form. Overall the proposed method was found to be suitable and accurate for the Quantitative determination of the drug in Tablet dosage form. The method was simple, precise, accurate and sensitive and applicable for the simultaneous determination of Rosuvastatin and Fenofibrate in bulk drug and in combined dosage forms.

Analysis of Veterinary Drug and Pesticide Residues Using the Ethyl Acetate Multiclass/Multiresidue Method in Milk by Liquid Chromatography-Tandem Mass Spectrometry.

A rapid and simple multiclass, ethyl acetate (EtOAc) multiresidue method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) detection was developed for the determination and quantification of 26 veterinary drugs and 187 total pesticide residues in milk. Sample preparation was a simple procedure based on liquid–liquid extraction with ethyl acetate containing 0.1% acetic acid, followed by centrifugation and evaporation of the supernatant. The residue was dissolved in ethyl acetate with 0.1% acetic acid and centrifuged prior to LC-MS/MS analysis. Chromatographic separation of analytes was performed on an Inertsil X-Terra C18 column with acetic acid in methanol and water gradient. The repeatability and reproducibility were in the range of 2 to 13% and 6 to 16%, respectively. The average recoveries ranged from 75 to 120% with the RSD (n = 18). The developed method was validated according to the criteria set in Commission Decision 2002/657/EC and SANTE/11945/2015. The validated methodology represents a fast and cheap alternative for the simultaneous analysis of veterinary drug and pesticide residues which can be easily extended to other compounds and matrices.

Development and Validation of RP-HPLC Method for Estimation of Quetiapine Fumarate in Pharmaceutical Formulations

Objective : A simple, rapid, accurate and precise RP-HPLC method was developed for the determination of Quetiapinefumarate in pure and tablet dosage forms. Materials and Methods : Separation of the drug was achieved on aisocratic Shimadzu prominence HPLC instrument on a Waters Xterra C18 column (250x4.6 mm, 5 μ). Results : The method showed a linear response for concentration in the range of 50–150 μg/mL using buffer (9.2 ± 0.05) and acetonitrile in the ratio of 51:49 v/v with detection at 254 nm with a flow rate of 1.0 mL/min and retention time was 6.588 min. Conclusion: The method was statistically validated for linearity, accuracy, precision and selectivity. Quantitative and recovery studies of the dosage form were also carried out and analyzed, the %RSD from recovery studies was found to be less than 1. Key words: Quetiapinefumarate, Isocratic, C 18 , HPLC, Tablets.

A sensitive liquid chromatography-mass spectrometry bioanalytical assay for a novel anticancer candidate--ZMC1.

ZMC1 {azetidinecarbothioic acid, [1-(2-pyridinyl) ethylidene] hydrazide} is a lead compound being developed as one of the first mutant p53 targeted anti-cancer drugs. Establishing a precise quantitative method is an integral component of this development. The aim of this study was to develop a sensitive LC/MS/MS assay suitable for assessing purity, stability and preclinical pharmacokinetic studies of ZMC1. Acetonitrile protein precipitation extraction was chosen for plasma sample preparation with satisfactory recovery (84.2-92.8%) for ZMC1. Chromatographic separation was achieved on an Xterra C18 column (50 × 4.6 mm, 3.5 µm) using a gradient elution with mobile phase of 0.1% formic acid in water and acetonitrile. ZMC1 and internal standard 2-amino-6-bromobenzothiazole were identified using selected-ion monitoring mode at m/z 235.2/178.2 and m/z 231.0/150.0 at retention times of 5.2 and 6.3 min, respectively. The method was validated with a linearity range of 3.9-500.0 ng/mL in human plasma and showed acceptable reproducibility with intra- and interday precisions <5.9 and 10.5%, and accuracy within ±5.4% of nominal values. This analytical method together with basic stability data in plasma and plasma binding experiments provides a reliable protocol for the study of ZMC1 pharmacokinetics. This will greatly facilitate the pre-clinical development of this novel anti-cancer drug.

Retention of [18F]fluoride on reversed phase HPLC columns

As [18F]fluoride is a starting reagent in the radiosynthesis of most fluorine-18 labeled positron emission tomography (PET) tracers, its chromatographic behavior on reversed phase (RP) HPLC columns is important for the purification performance and accuracy of RP HPLC quality control methods. We have investigated the chromatographic behavior and recovery of [18F]fluoride as a function of the type and brand of RP HPLC column, the pH and the composition of the mobile phase. Elution and elution profile of [18F]fluoride from six RP-HPLC columns (Waters XBridge C18 3mm×100mm 3.5μm; Grace Platinum EPS C18 4.6mm×100mm, 3μm; Waters XTerra C18 4.6mm×250mm, 5μm; Phenomenex C18 4.6mm×150mm, 5μm; Hamilton PRP-1 column 4.1mm×150mm, 5μm; Merck KGaA Chromolith Performance C18 3mm×100mm) eluted with mobile phase composed of phosphate or acetate buffers (pH 2, 3, 4, 5, 7.3 and 9) and acetonitrile or ethanol as organic modifier were characterized. The elution profile was determined by on-line radioactivity measurement in the column eluate and recovery was calculated by comparison of radioactivity eluted with the HPLC column present or absent in the chromatographic flow path. Interestingly, [18F]fluoride recovery increased with increasing pH. At pH 3 all packed silica-based columns showed significant retention of fluorine-18, whereas almost no retention was observed on a polymeric PRP-1 column. However at pH 5, [18F]fluoride recovery was above 90% for each tested column. In addition, small differences were observed when changing the composition of the mobile phase. We therefore recommend to use a mobile phase with pH>5 for silica based C18 columns for both quality control and semi-preparative HPLC of fluorine-18 labeled PET radiopharmaceuticals. If required a lower pH can be used in combination with a polymer based HPLC column.

Selective separation and characterization of the stress degradation products of ondansetron hydrochloride by liquid chromatography with quadrupole time-of-flight mass spectrometry.

Ondansetron hydrochloride was subjected to forced degradation studies under various conditions of hydrolysis (acidic, basic, and neutral), oxidation, photolysis, and thermal as prescribed by International Conference on Harmonisation guideline Q1A (R2). A simple, selective, precise, and accurate high-performance liquid chromatography method was developed on a Waters Xterra C18 (150 × 4.6 mm id, 3.5 μm) column using 10 mM ammonium formate (pH 3.0)/methanol as a mobile phase in gradient elution mode at a flow rate of 0.6 mL/min. The method was extended to liquid chromatography quadrupole time-of-flight tandem mass spectrometry for identification and structural characterization of stress degradation products of ondansetron. The drug showed significant degradation in base hydrolytic and photolytic stress conditions in the liquid state, while it was found to be stable in neutral, acidic, thermal, and oxidative stress conditions. A total of five degradation products were characterized and most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M + H](+) ions of the drug and its degradation products. Finally, the developed method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonisation guideline Q2 (R1).

New Validated Stability Indicating RP-HPLC Method for Simultaneous Estimation of Metformin and Alogliptin in Human Plasma

A validated new stability indicating RP-HPLC method for the quantitative determination of metformin and alogliptin in human plasma was developed as per US-FDA guidelines. The drug was spiked in the plasma and extracted with mobile phase by precipitation method. The extracted analyte was injected into X-Terra C18 (4.6 × 150 mm, 3.5 μm, Make: ACE) or equivalent, maintained at 25°C temperature and effluent was monitored at 235 nm. The mobile phase was consisted of sodium dihydrogen ortho phosphate [pH 4.0]:acetonitrile [HPLC Grade] (70:30 v/v). The flow rate was maintained at 1.0 mL/min. The calibration curve for metformin and alogliptin was linear from 300.0 to 700.0 μg/mL (r2=0.997) and 7.5 to 17.5 μg/mL (r2=0.998) respectively. The inter-day and intra-day precision was found to be within limits. The Lower limit of quantification (LLOQ) for metformin and alogliptin were 5.936 and 1.983 μg/mL respectively. The average % recovery for metformin and alogliptin were 100.17 and 99.40-99.55% respectively and reproducibility was found to be satisfactory. This RP-HPLC method is suitable for determining the concentration of metformin and alogliptin in human plasma and it can applied for routine analysis for determination of the metformin and alogliptin from dosage form during pharmacokinetic study.

Double Off-line Two-dimensional Liquid Chromatography for Separation and Identification of Compounds in Salvia Miltiorrhiza (Danshen)

Background: Danshen is an important traditional Chinese medicine (TCM) used for the treatment of cardiovascular and cerebrovascular diseases. Separation and analysis of its components have been widely investigated. However, the systematical two dimensional liquid chromatography (2D-LC) methods have not been developed to comprehensively separate and characterize its components. Objective: In this work, double off-line 2D-LC methods were aimed to develop for the systematical separation of compounds from Danshen. Methods: Using solid phase extraction (SPE), the Danshen extract was divided into a medium-polar fraction (Sample I) and a weak-polar fraction (Sample II) according to their polarities. Based on reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) modes, a 2D-HILIC × RPLC system and a 2D-RPLC × RPLC system were designed for the separation of Sample I and Sample II, respectively. According to reversed-phase and HILIC columns selectivities characterized in our previous reports, ZIC-HILIC and XTerra C18 were employed to build the 2D-HILIC × RPLC system and Click TE-CD and XTerra C18 for the 2D-RPLC × RPLC system, respectively. Results: The 2D-HILIC × RPLC and 2D-RPLC × RPLC systems exhibited excellent orthogonality for the separation of Sample I and Sample II, respectively. Their orthogonalities were 88.42% and 63.24%. Based on these double 2D-LC systems combined with mass spectrometry, at least 200 compounds were found and 33 compounds of them were identified, including 16 phenolic acids and 17 diterpenoid quinines. Conclusion: These results suggest that these two off-line 2D-LC methods are effective for the separation and characterization of components in Danshen.

DoE Approach: A Stability Indicating RP-HPLC Method for Simultaneous Estimation of Methylparaben, Mometasone furoate and Eberconazole nitrate in Topical Formulations

A rapid and sensitive stability indicating RP-HPLC method is developed for the simultaneous estimation of Methylparaben, Mometasone Furoate and Eberconazole Nitrate in topical formulations. Chromatographic separation was achieved on Waters Xterra C18 (150 × 4.6 mm, 5µm) using a mobile phase constituted of water and methanol (35:65, v/v) at a flow rate of 1.50 mL/min and column temperature of 30˚C. All three components were measured with uv detection at 235 nm. Force degradation study was conducted to determine Methylparaben, Mometasone Furoate and Eberconazole Nitrate in the presence of degradants and excipients peaks. Developed method was validated for method precision, specificity, linearity, accuracy, robustness and solution stability as per ICH guidelines. Method is showing linearity in the range of 0.25-188, 0.50-75 and 2.0-750 µg/mL for Methylparaben, Mometasone Furoate and Eberconazole Nitrate respectively. The method was proved to be robust by conducting DOE study. The method is suitable for stability studies, routine analysis and quality control of topical formulations containing these components, either alone or in combination.

Structural characterization of alkaline and oxidative stressed degradation products of lurasidone using LC/ESI/QTOF/MS/MS

A selective, accurate, precise and robust stability indicating liquid chromatography assay method was developed for the monitoring of a novel antipsychotic drug, lurasidone, in the presence of its degradation products (DPs). Also, we investigated degradation behavior of the drug under various stressed conditions such as hydrolytic (acidic, basic and neutral), oxidation, photolytic and thermal. The drug was found to be degraded under base hydrolytic and oxidative conditions, while it was stable in acid and neutral hydrolytic, photolytic and thermal conditions. The method showed adequate separation of lurasidone and its DPs on Xterra C18 (150mm×4.6mm i.d., 3.5μm) column using 20mM ammonium formate (pH 3.0): acetonitrile as a mobile phase in gradient elution mode at a flow rate of 0.6mL/min. This method was extended to liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI/QTOF/MS/MS) for structural characterization of DPs. A total of five DPs were characterized by LC/ESI/QTOF/MS/MS studies. Most probable mechanisms for the formation of DPs were proposed. The developed method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonization Guideline Q2 (R1).