Stability-Indicating UPLC Method for the Determination of Bisoprolol Fumarate and Hydrochlorothiazide: Application to Dosage Forms and Biological Sample

A sensitive, selective and accurate ultra performance liquid chromatographic method has been developed and validated for the simultaneous determination of bisoprolol fumarate and hydrochlorothiazide in their combined dosage forms and as well as in spiked human urine samples. The separation was achieved on an Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column, at 40 °C with mobile phase consisting of acetonitrile:phosphate buffer (20 mM) at pH 3.0 with a gradient elution at 225 nm. Bisoprolol fumarate and hydrochlorothiazide were well separated in <1.5 min with good resolution and without any tailing and interference of excipients. The method was fully validated according to ICH guidelines in terms of accuracy, precision, linearity and specificity. A linear response was observed over the concentration range 0.5–150 μg mL^?1 for hydrochlorothiazide and 0.5–250 μg mL^?1 for bisoprolol fumarate. Limit of detection and limit of quantitation for hydrochlorothiazide were calculated as 0.01 and 0.03 μg mL^?1, respectively, and for bisoprolol fumarate were 0.07 and 0.21 μg mL^?1, respectively. Moreover, bisoprolol fumarate and hydrochlorothiazide were subjected to degradation conditions such as hydrolytic, oxidative and thermal stress conditions to evaluate the ability of the proposed method for the separation of bisoprolol fumarate and hydrochlorothiazide from their degradation compounds.     

On-Line Two-Dimension Liquid Chromatography for the Analysis of Ingredients in the Medicinal Preparation of Coptis Chinensis Franch

An on-line two-dimension microflow liquid chromatography was developed for better separation and analysis of the highly complex ingredients of medicinal preparation of traditional Chinese medicine Coptis Chinensis Franch. A two-valve switching system was utilized for two-dimension chromatography with strong cation exchange and reverse-phase capillary columns separation. The components were separated well by this system and yielded over 420 peaks. Under the optimal condition, 4 compounds were detected quantitatively. A good linear relationship was obtained from 0.2 µg mL^?1 to 24 µg mL^?1with detection limits (S/N = 3) ranging from 0.05 µg mL^?1 to 0.2 µg mL^?1for the compounds. We demonstrated that the method can be successfully applied to the analysis of a natural complex sample, with satisfactory results.     

A Stability-Indicating Assay of Brimonidine Tartrate Ophthalmic Solution and Stress Testing Using HILIC

A stability-indicating hydrophilic interaction liquid chromatography (HILIC) method has been developed and validated for the quantitative determination of Brimonidine tartrate (BT) formulated as an ophthalmic solution. Isocratic separation was achieved using an acetonitrile-buffer mixture (92:8, v/v) at pH 7.1 on an unmodified silica column (250 × 4.6 mm, 5 μm). The drug was subjected to oxidative, hydrolytic, photolytic and thermal stress conditions and complete separation was achieved for the parent compound and degradation products. The influence of acetonitrile, pH and ionic strength of the buffer was studied. Linearity range and recoveries for BT were 100–400 μg mL^?1 and 100.12%, respectively. The method was validated for BT and indicated that the method was sufficiently sensitive with a limit of detection at 0.005 μg mL^?1 and a limit of quantitation at 0.02 μg mL^?1, respectively.     

Simultaneous Determination of Amoxicillin and Clavulanic Acid in Dog Plasma by High-Performance Liquid Chromatography Tandem Mass Spectrometry

A rapid, sensitive, and specific high-performance liquid chromatography tandem mass spectrometric method was developed for the simultaneous determination and confirmation of amoxicillin and clavulanic acid in plasma. Plasma sample was subjected to a simple deproteinization with acetonitrile, and then the supernatant was directly diluted by water. Analysis was performed on a Phenomenex Luna C₈ reversed-phase column by detection with mass spectrometry in negative ions multiple reaction monitoring mode. A gradient elution program with 0.1% formic acid and acetonitrile was performed at a flow of 0.25 mL min^?1. There is good linearity in the range of 0.5–500 ng mL^?1 for both amoxicillin and clavulanic acid. The decision limits of amoxicillin and clavulanic acid were 0.06 ng mL^?1 and 0.08 ng mL^?1 in plasma, respectively, and the detection capabilities of two analytes were below 0.5 ng mL^?1. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The extraction recoveries of amoxicillin and clavulanic acid were between 102% and 115% in plasma at three spiked levels of 0.5, 50, and 500 ng mL^?1, with the relative standard deviations less than 15% for each analyte. The developed method was applied to pharmacokinetic studies of amoxicillin and clavulanic acid tablets in healthy beagles.     

Development and Validation of a Novel Gradient LC Method for Simultaneous Determination of Isoniazid and Acetylisoniazid in Human Plasma

The goal of this study was to develop and validate a new gradient high-performance liquid chromatography method for the simultaneous determination of isoniazid (INH) and acetylisoniazid (Ac-INH) in human plasma samples. A C18 reversed-phase column was employed for separation followed by UV detection at 266 nm. The calibration involved the use of five concentration levels ranging from 1 to 20 μg mL^?1 for both analytes. The developed method was validated using ICH guidelines. The calibration curve was found to be linear with correlation coefficient values (r ²) above 0.9991 and the highest RSD% values for intra-day assays were found to be 6.34 and 2.57% for INH and Ac-INH, respectively. The highest RSD% values for inter-day assays were 9.31 and 10.17% for INH and Ac-INH, respectively. LOD was calculated to be 0.1 and 0.15 μg mL^?1 for INH and Ac-INH, respectively. LOQ was calculated to be 0.33 and 0.5 μg mL^?1 for INH and Ac-INH, respectively.     

Determination of Atenolol in Human Plasma by Pseudo Reversed Phase Liquid Chromatography-Tandem Mass Spectrometry

Previous HPLC determination of atenolol on reversed-phase packings has often required a mobile phase containing three components: organic modifier, buffer and ion-pairing reagent or organic amine. In addition to the complexity of the eluents employed, alkyl sulphonates and organic amines in the mobile phase can reduce the life of silica-based bonded columns. A new simple, rapid and sensitive method—pseudo reversed-phase liquid chromatography/tandem mass spectrometry has been developed for the analysis of atenolol in human plasma using bare silica as the stationary phase coupled with a simple mobile phase consisted of 5% acetonitrile and 95% formate buffer. The optimization of separation is fast and easy. The assay was validated for the concentration range 1–100 ng mL^?1 with a detection limit of 1 ng mL^?1. Moreover, the silica column was durable with the mainly aqueous eluents. No obvious loss in performance was observed for 30,000 column volumes of eluent.     

Development and Validation of a Stability-Indicating LC Method for the Determination of Rupatadine in Pharmaceutical Formulations

A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of rupatadine in pharmaceutical dosage forms. The LC method was carried out on a Gemini C₁₈ column (150 mm × 4.6 mm I.D.), maintained at 30 °C. The mobile phase consisted of ammonium acetate buffer (pH 3.0; 0.01 M) with 0.05% of 1-heptanesulfonic acid–acetonitrile (71.5:28.5, v/v), run at a flow rate of 1.0 mL min^?1 and using photodiode array (PDA) detection at 242 nm. The chromatographic separation was obtained with retention time of 5.15 min, and was linear in the range of 0.5–400 μg mL^?1 (r ² = 0.9999). The specificity and stability-indicating capability of the method was proven through the degradation studies and showing also, that there was no interference of the excipients. The accuracy was 100.39% with bias lower than 0.58%. The limits of detection and quantitation were 0.01 and 0.5 μg mL^?1, respectively. Moreover, method validation demonstrated acceptable results for precision, sensitivity and robustness. The proposed method was applied for the analysis of pharmaceutical dosage forms assuring the therapeutic efficacy.     

Development and Validation of a RP-HPLC–PDA Method for Determination of Curcuminoids in Microemulsions

A sensitive, specific and rapid high-performance liquid chromatography method was developed in an effort to quantify extremely low curcuminoid levels for the future transdermal experiments where the curcuminoids are incorporated with excipients such as microemulsion, liposomes, and micelles. The chromatographic separation was performed using a Symmetry^® C₁₈, 250 × 4.6 mm, 5-μm column, with a mobile phase composed of 5 mM acetonitrile:phosphoric acid (45:55, v/v) at a flow rate of 1.0 mL min^?1, it was sensitive with a low limit of quantitation for curcuminoids (0.626 ng mL^?1 for curcumin) and good linearity (r ² ≥ 0.999) over the range 1–100 ng mL^?1. All the validation data, such as accuracy and precision, were within the required limits from the ICH guideline. The assay method was successfully applied during forced degradation of curcuminoid solutions. The method retained its accuracy and precision when the standard addition technique was applied.     

Determination of Gastrodin and Ligustrazine Hydrochloride in Plasma and Brain Dialysate by LC–Tandem MS

A gradient liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of gastrodin and ligustrazine hydrochloride in rat plasma and brain dialysates. Zolpidem was used as internal standard. For plasma samples, solid-phase extraction was used and the brain dialysates were collected from freely moving rats using brain microdialysis. Both were followed by HPLC separation and positive electrospray ionization tandem mass spectrometry detection (ESI–MS–MS). Chromatographic separation was achieved on a Symmetry RP-18 column using gradient elution with methanol and water containing 0.5% formic acid and 2 mM ammonium formate. Selected reaction monitoring (SRM) mode was used for quantitation. Good linearities were obtained in the range of 0.05–100 and 0.01–50 μg mL^?1 for gastrodin and ligustrazine hydrochloride in rat plasma, and 0.05–1,000 ng mL^?1 for both in dialysate. The lower limit of quantitation was 0.01 ng mL^?1 for gastrodin and 0.05 ng mL^?1 for ligustrazine. The method is precise and reliable and can be applied to pharmacokinetic studies.     

Development and Validation of an LC Method for Simultaneous Determination of Ascorbic Acid and Three Phenolic Acids in Sustained Release Tablets at Single Wavelength

A simple, rapid and sensitive column liquid chromatographic method was developed and validated to measure simultaneously the amount of ascorbic acid and phenolic acids at single wavelength (240 nm) in order to assess drug release profiles and drug-excipients compatibility studies for a new sustained release tablet formulation and its subsequent stability studies. A combined isocratic and linear gradient reversed-phase LC method was carried out at 240 nm. Quantification was achieved with reference to the external standards. The linearity for concentrations between 0.042 and 0.150 mg mL^?1 for ascorbic acid, 0.084–0.250 mg mL^?1 for chlorogenic acid, 0.053–0.360 mg mL^?1 for caffeic acid, and 0.016–0.250 mg mL^?1 for ferulic acid (r > 0.99 for all analytes) were established. The recovery of the active ingredients from the samples was at the range of 92.3–102.9%. Intra- and inter-day precisions were less than 2.5%. The limits of detection and quantification were 8 and 24 μg mL^?1 for ascorbic acid, 18 and 54 μg mL^?1 for chlorogenic acid, 37 and 112 μg mL^?1 for caffeic acid, and 11 and 34 μg mL^?1 for ferulic acid. The determination of the four active ingredients was not interfered by the excipients of the products. Samples were stable in the release mediums (37 °C) at least for 12 h.     

Determination of Abacavir, Lamivudine and Zidovudine in Pharmaceutical Tablets, Human Serum and in Drug Dissolution Studies by HPLC

A simple, accurate, precise and fully automated method for the simultaneous determination of abacavir, lamivudine and zidovudine in pharmaceutical tablets, human serum samples and drug dissolution studies has been developed. Separation was performed on a 5 μm Zorbax^® C₁₈ column (150 × 4.6 mm ID) with methanol:water:phosphate buffer at pH 5.65 (80:10:10; v/v/v) isocratic elution in less than 7 min with a flow rate of 0.6 mL min^?1.Good sensitivity for all analytes was observed with UV detection at 275 nm. The method allowed quantitation over the 500–3,000 ng mL^?1 range for abacavir and 500–5,000 ng mL^?1 range for lamivudine and zidovudine. The method has been applied, without any interference from excipients or endogenous substances, for the simultaneous determination of these three compounds in tablets. Human serum and drug dissolution studies.     

Simultaneous Determination of Ibuprofen and Diphenhydramine Citrate in Tablets by Validated LC

A stability-indicating LC method was developed for the simultaneous determination of ibuprofen and diphenhydramine citrate in pharmaceutical dosage forms. The chromatographic separation was achieved on an Inertsil ODS 3V, 150 × 4.6 mm, 5 μm, column. The mobile phase contained a mixture of 50 mM potassium dihydrogen phosphate buffer:acetonitrile:triethylamine:glacial acetic acid (55:45:0.2:0.2, v/v/v/v). This method allowed the determination of 2.85–9.14 mg mL^?1 of ibuprofen and 0.54–1.73 mg mL^?1 of diphenhydramine citrate, in a diluent consisting of pH 7.2, 50 mM potassium dihydrogen phosphate buffer:acetonitrile (40:60, v/v). The flow rate was 1.2 mL min^?1 and the detection wavelength was 260 nm. The limit of detection for ibuprofen and diphenhydramine citrate was 1.72 and 0.54 μg mL^?1 and the limit of quantification was 5.73 and 1.64 μg mL^?1, respectively. This method was validated for accuracy, precision and linearity. The method was also found to be stability indicating.     

CZE Separation of New Drugs for Treatment of Leukemia

Imatinib, bosutinib, dasatinib, pazopanib, erlotinib, canertinib and vatalanib are new developed anticancer drugs, especially for treatment of leukemia. In this article, a fast and high throughput capillary zone electrophoresis method has been developed and validated for analysis of these new drugs in pharmaceutical formulas. The method can be easily utilized for determination of all the drugs in one run what is advantageous for the quality control in pharmaceutical industry because there is no need for changing and optimization of separation conditions when changing the analyte. The separation was performed using an uncoated fused silica capillary with 100 mmol L^?1 sodium phosphate buffer pH 2.75, voltage of 25 kV, hydrodynamic injection time of 5 s by 50 mbar, and detection at 214 nm. Under these conditions, the analysis took about 8 min. The validation of all the drugs resulted in recoveries in the range of 84–100 %. The method showed to be precise for all the drugs with RSDs of migration times lower than 0.9 % (interday precision). A very good linearity in the validated range (5–100 μg mL^?1) and the limits of detection (LODs) in the range of 0.5–2.0 (μg mL^?1) were achieved. Finally, we proved that the method is robust by the Youden’s test. Therefore, our method can be successfully applied for analysis of the real pharmaceutical samples.     

Determination of Danofloxacin and Marbofloxacin in Milk Samples by Micellar Liquid Chromatography with Fluorescence Detection

Abstract

A simple, rapid, and sensitive analytical method has been developed for the determination of two fluoroquinolones, danofloxacin and marbofloxacin, in bovine milk samples. Separation and quantification were performed by micellar liquid chromatography with fluorescence detection (MLC?FD), using sodium dodecyl sulfate (SDS) as a surfactant. The influence of the principal factors, namely, the micelle concentration, the amount of organic modifier, tail‐reducing agents, the pH, and the temperature were studied. The suitable condition was found to be 75 mM SDS?10 mM phosphate buffer–18 mM tetrabutylammonium bromide/3% (v/v) 1‐propanol at pH 3.0 for the separation of marbofloxacin, danofloxacin, and tosufloxacin (internal standard) in about 20 min. The linear concentration range of application was 1.8–30.0 ng · mL^?1 for danofloxacin and 16–120 ng · mL^?1 for marbofloxacin, and the relative standard deviation ranged between 4.9 and 2.7%. The limit of detection found for danofloxacin was 0.5 ng · mL^?1 and 5 ng · mL^?1 for marbofloxacin. These values were lower than the maximum residue limits (MRLs) established by the European Union for these compounds in bovine milk. It was applied to check the eventual existence of these compounds above these limits on commercial milk samples. The validation method was completed with spiked milk samples. Recovery levels obtained were 90.3–108.2%.     

Determination of Active Ingredients of Origanum Vulgare L. and Its Medicinal Preparations by Capillary Electrophoresis with Electrochemical Detection

Abstract

A method based on capillary electrophoresis with electrochemical detection (CE‐ED) has been developed for the first time for the separation and determination of isovanillic acid, vanillic acid, quercetin, rosmarinic acid, caffeic acid, and protocatechuic acid in Origanum vulgare L. and its medicinal preparations. The effects of working electrode potential, pH level, concentration of running buffer, separation voltage, and injection time on CE‐ED were investigated. Under the optimum conditions, the analytes could be separated in a 50 mmol L^?1 borate buffer (pH 8.7) within 21 min. A 300‐µm diameter carbon disk electrode has a good response at +0.95 V (vs. SCE) for all analytes. The response was linear over three orders of magnitude with detection limits (S/N=3) ranging from 4×10^?8 g mL^?1 to 2×10^?7 g mL^?1 for the analytes. The method has been successfully applied to the analysis of real sample, with satisfactory results.     

Rapid Determination of Water-Soluble and Fat-Soluble Vitamins in Commercial Formulations by MEEKC

Microemulsion electrokinetic capillary chromatography has been successfully applied to the separation and determination of water-soluble vitamins (thiamine hydrochloride, riboflavin, niacin, pyridoxine hydrochloride, folic acid, cobalamin, ascorbic acid) and a fat-soluble vitamin (α-tocopherol acetate). The optimal microemulsion buffer contained sodium dodecylsulfate (SDS) as surfactant, butan-1-ol as the co-surfactant, ethyl acetate as the oil and pH 9.2 tetraborate buffer, modified with 15% (v/v) 2-propanol. UV detection at 214 nm gave adequate sensitivity without interference from sample excipients. Under the optimized conditions, the vitamins were baseline separated in less than 7 min. Analytical curves of peak area versus concentration presented coefficients of determination (R ² ) > 0.99, acceptable limits of quantification between 8.40 and 16.23 μg mL^?1 were obtained. Vitamin levels in liquid formulation were quantified with intra-day precision better than 0.99% RSD for migration time and 1.19% RSD for peak area ratio. Recoveries ranged between 98.7 and 101.7%. The method was considered appropriate for rapid and routine analysis.     

A Simple and Rapid CZE Determination of Tiapride Hydrochloride and Related Impurities in Pharmaceutical Formulations

A simple, fast, inexpensive capillary zone electrophoresis method for the separation and determination of tiapride hydrochloride and its two related impurities in pharmaceutical formulations has been developed and validated. The successful separation of these compounds was achieved in less than 3 min using a fused silica capillary and photodiode array detector at 218 nm. The best conditions were obtained using a 10 mM sodium tetraborate (pH 8.0) as the running buffer. The linear responses covered the ranges from 1.0 to 100 μg mL^?1 (R = 0.9989) for tiapride hydrochloride. The detection (LOD) and quantitation limits (LOQ) for tiapride hydrochloride were 2.7 and 9.0 μg mL^?1, respectively. The intra- and inter-day relative standard deviations for migration times and peak areas were less than 0.47 and 5.7%, respectively. The method was validated for the determination of tiapride hydrochloride in commercial tablets.     

Enantioselective determination of modafinil in pharmaceutical formulations by capillary electrophoresis, and computational calculation of their inclusion complexes

A fast capillary electrophoretic method is described for the separation and determination of the enantiomers of the novel wake-promoting agent, modafinil. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, buffer concentration, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 5 min with resolution factor Rs?=?2.51, using a bare fused-silica capillary and a background electrolyte (BGE) of 25 mM H₃PO₄?1 M tris solution; pH 8.0; containing 30 mg mL^?1 of sulfated-β-cyclodextrin (S-β-CD). The separation was carried out in normal polarity mode at 25 ^?C, 18 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy were included. The developed method was successfully applied to the assay of enantiomers of modafinil in pharmaceutical formulations. The computational calculations for the enantiomeric inclusion complexes rationalized the reasons for the different migration times between the modafinil enantiomers.     

HPLC Determination of Letrozole in Plasma Using Fluorescence Detection: Application to Pharmacokinetic Studies

A simple, rapid and sensitive HPLC method has been developed and validated for the analysis of letrozole in human plasma. The separation was achieved on a monolithic silica column using acetonitrile–phosphate buffer. A fluorescence detector was used for the quantitation with excitation and emission wavelengths at 230 and 295 nm. The assay enables the measurement of letrozole for therapeutic drug monitoring with a minimum quantification limit (LOQ) of 0.5 ng mL^?1. The method involves a simple, one-step extraction procedure with complete recovery. Calibration was linear over the concentration range 0.5–80 ng mL^?1. The coefficients of variation for inter-day and intra-day assay were found to be less than 8%.     

LC–MS–MS Analysis of Strictosamide in Rat Plasma, and Application of the Method to a Pharmacokinetic Study

A rapid and simple high-performance liquid chromatographic tandem mass spectrometric method has been developed and validated for analysis of strictosamide in rat plasma. Chromatographic separation was achieved on a C₁₈ column by gradient elution with mixtures of methanol, water, and acetonitrile containing 0.05% acetic acid. Digoxin was used as internal standard. Selected reaction monitoring (SRM) was used for MS quantitation. Linearity was good in the range 0.05–20 ng mL^?1 in rat plasma. The lower limit of quantitation was 0.04 ng mL^?1. The method is precise and reliable and can be applied to pharmacokinetic studies.