Single robust RP-HPLC analytical method for quantification of curcuminoids in commercial turmeric products,Ayurvedic medicines,and nanovesicular systems

A single robust reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated as per International Conference on Harmonization guidelines for the accurate quantification of curcuminoids in commercial turmeric products, Ayurvedic medicines, and nanovesicular systems. The proposed chromatographic method was found to be specific, linear (r²?≥?0.999), precise at intra- and inter-day levels (percentage relative standard deviation <2.0%), accurate (percentage recovery 99.14–102.29%), and robust. The limits of detection and quantification were found to be 7.40 and 24.70?ng?mL^?1 for curcumin, 9.24 and 30.80?ng?mL^?1 for demethoxycurcumin, and 6.48 and 21.61?ng?mL^?1 for bisdemethoxycurcumin, respectively. Among different commercial turmeric products and Ayurvedic medicines tested, the contents of curcumin (3.54?±?0.06–25.8?±?0.08?mg?g^?1), demethoxycurcumin (1.28?±?0.02–9.97?±?0.03?mg?g^?1), and bisdemethoxycurcumin (0.50?±?0.01–5.97?±?0.01?mg?g^?1) varied significantly. The developed method was effectively applied to the determination of encapsulation efficiency of curcuminoids (ranged between 84.33?±?3.50 and 96.59?±?2.53%) in the nanovesicular systems. In conclusion, the reported method is suitable for the analysis of curcuminoids in a wide variety of turmeric products and used for the quality control of products that contain curcuminoids.     

GC Determination of Prilocaine HCl in Human Plasma: Analytical Application to Real Samples

A gas chromatographic (GC) method with a rapid and simple sample preparation was developed and validated for determination of prilocaine in human plasma. The validation parameters of linearity, precision, accuracy, recovery, specificity, limit of detection and limit of quantification were studied. The range of quantification for the GC method was 50–300 ng mL^?1 in plasma. Intra- and inter-day precision, expressed as the relative standard deviation (RSD) were less than 4.5%, and accuracy (relative error) was better than 8.0% (n = 6). The analytical recovery of prilocaine HCl from plasma has averaged 96.5% and the recovery of internal standard (lidocaine HCl) reached 96.8%. The limit of quantification (LOQ) and the limit of detection (LOD) of the method for plasma were 50 and 40 ng mL^?1, respectively. Also the developed and validated method was applied to three healthy volunteers to whom a local anaesthesia with citanest was administered.     

LC Determination of Lidocaine and Prilocaine Containing Potential Risky Impurities and Application to Pharmaceuticals

A liquid chromatographic method for the determination of lidocaine (LID), prilocaine (PRL) and their impurities 2,6-dimethylaniline (DMA) and o-toluidine (TOL) has been developed. The analysis was performed on a reversed phase C18 Hypersil BDS column at ambient temperature. A mobile phase consisting of Briton-Robinson buffer, pH 7—methanol—acetonitrile (40: 45: 15 v/v/v) was used at a flow rate of 1.2 mL min^?1. Detection was achieved at 225 nm using benzophenone as internal standard over the concentration range 1.25–80 μg mL^?1 for all analytes. The relative standard deviations RSD (n = 7) for the assay were less than 0.95%. Limit of detection values were found to be 0.346, 0.423, 0.112 and 0.241 μg mL^?1 for LID, PRL, DMA and TOL, respectively. The intraday and the inter-days RSD % indicated the precision of the procedure. The method proved to be suitable for the quality control of LID and PRL in pharmaceuticals.     

Optimisation of an in-tube solid phase microextraction method coupled with HPLC for determination of some oestrogens in environmental liquid samples using different capillary columns

A simple on-line method for simultaneous determination of some oestrogens including oestriol (E3), norethisterone (NORE), ethynylestradiol (EE2), D-norgestrel (NORG) and bisphenol A (BPA), in environmental liquid samples was developed by coupling in-tube solid phase microextraction (in-tube SPME) to high-performance liquid chromatography with diode array (DAD) and fluorescence (FLD) detectors. Two capillary chromatographic columns (Supel-Q? and Carboxen? 1006 porous layer open tubular) were selected to develop this method. To achieve optimum extraction performance, several parameters were investigated including number of draw/eject cycles and the sample volume for each of the columns. Reproducibility was satisfactory for inter- and intra-day precision, yielding % RSDs of less than 10% and 7.6%, respectively. Limits of detection (LODs) and quantification (LOQs) for the proposed method using a DAD detector were achieved in the ranges of 0.04–0.63?ng?mL^?1 and 0.12–1.9?ng?mL^?1, depending of the capillary column used. Fluorescence detection improved these parameters for E3, BPA and EE2, obtaining LODs of 0.005–0.03?ng?mL^?1 and LOQs of 0.015–0.08?ng?mL^?1 using Supel-Q and LODs of 0.01–0.015?ng?mL^?1 and LOQs of 0.025–0.04?ng?mL^?1 using Carboxen. The proposed method was successfully applied to spiked environmental waters obtaining recoveries greater than 80%.     

A Stability-Indicating LC Method for Analysis of Balsalazide Disodium in the Bulk Drug and in Pharmaceutical Dosage Forms

A novel, sensitive, stability-indicating gradient RP-LC method has been developed for quantitative analysis of balsalazide disodium and its related impurities both in the bulk drug and in pharmaceutical dosage forms. Efficient chromatographic separation was achieved on a C₁₈ stationary phase with a simple mobile-phase gradient prepared from methanol and phosphate buffer (10 mm potassium dihydrogen orthophosphate monohydrate, adjusted to pH 2.5 by addition of orthophosphoric acid). The mobile-phase flow rate was 1.0 mL min^?1. Quantification was achieved by use of ultraviolet detection at 240 nm. Under these conditions resolution of balsalazide disodium from its three potential impurities was greater than 2.0. Regression analysis resulted in a correlation coefficient greater than 0.99 for balsalazide disodium and all three impurities. This method was capable of detecting the three impurities at 0.003% of the test concentration of 0.3 mg mL^?1, using an injection volume of 10 μL. Inter-day and intra-day precision for all three impurities and for balsalazide disodium was within 2.0% RSD. Recovery of balsalazide disodium from the bulk drug (99.2–101.5%) and from pharmaceutical dosage forms (99.8–101.3%), and recovery of the three impurities (99.1–102.1%) was consistently good. The test solution was found to be stable in 70:30 (v/v) methanol–water for 48 h. When the drug was subjected to hydrolytic, oxidative, photolytic, and thermal stress, acidic and alkaline hydrolysis and oxidizing conditions led to substantial degradation. The RP-LC method was validated for linearity, accuracy, precision, and robustness.     

Development and Validation of a New Analytical Method for the Determination of Related Components and Assay of Ranolazine in Bulk Drug and Pharmaceutical Dosage Forms by LC

A novel liquid chromatographic method has been developed and validated for the determination of ranolazine, its potential four impurities in drug substance and drug products. Efficient chromatographic separation was achieved on a C18 stationary phase (150 × 4.6 mm, 3.0 microns particles) with simple mobile phase combination delivered in gradient mode at a flow rate of 1.0 mL min^?1 at 210 nm. In the developed method, the resolution between ranolazine and its four potential impurities was found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) greater than 0.999 for ranolazine and for its four impurities. This method was capable to detect all four impurities of ranolazine at a level below 0.004% with respect to test concentration of 1.0 mg mL^?1 for a 10 μL injection volume. The method has shown good, consistent recoveries for ranolazine (98.8–101.1%) and for its four impurities (97.2–100.3). The test solution was found to be stable in the diluent for 48 h. The drug was subjected to stress conditions. The mass balance was found close to 99.5%.     

A Stability Indicating LC Method for Vardenafil HCl

A simple, sensitive gradient RP-LC assay method has been developed for the quantitative determination of vardenafil HCl in bulk drug and in pharmaceutical dosage forms, used to treat erectile dysfunction. The developed method is also applicable for the related substances determination. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in a gradient mode and quantification was carried out using ultraviolet detection at a flow rate of 1.0 mL min^?1. In the developed LC method the resolution between vardenafil and its four potential impurities was found to be greater than 3.0. Regression analysis shows an r ² value (correlation coefficient) greater than 0.99 for vardenafil and its four impurities. This method was capable of detecting all four impurities of vardenafil at a level of 0.009% with respect to test concentration of 1.0 mg mL^?1 for a 10 μL injection volume. The method has shown good and consistent recoveries for vardenafil (98.4–100.6%) and its four impurities (93.5–106.2%). The test solution was found to be stable in the diluent for 48 h. Mass balance was found close to 99.4%.     

Identification of Acepromazine and Its Metabolites in Horse Plasma and Urine by LC–MS/MS and Accurate Mass Measurement

Acepromazine maleate (Sedalin^?) was administered orally to six thoroughbred horses at a dose of 0.15?mg?kg^?1. Urine and blood samples were collected up to 412?h post-administration. Plasma and urine were hydrolysed; plasma samples were then processed using liquid–liquid extraction and urine samples using solid-phase extraction. A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification for acepromazine of 10?pg?mL^?1 in plasma and 100?pg?mL^?1 in urine. Acepromazine, hydroxyethylpromazine, hydroxyacepromazine, hydroxyethylpromazine sulphoxide, hydroxyethylhydroxypromazine, dihydroxyacepromazine and dihydroxyhydroxyethylpromazine were detected in the post-administration samples. The parent drug and its metabolites were identified using a combination of UPLC–MS/MS and accurate mass measurement. Separation of the structural isomers hydroxyethylpromazine sulphoxide and hydroxyethylhydroxypromazine was another significant outcome of this work and demonstrated the advantages to be gained from investing in chromatographic method development.     

Development and Validation of an Stability Indicating RP-LC Method for Determination of Imatinib Mesylate

A new, sensitive and stability indicating liquid chromatographic method has been developed for the determination of imatinib mesylate (IM). Efficient chromatographic separation was achieved using a C₁₈ column with simple mobile phase combination delivered in an isocratic mode and quantitation was carried out using ultraviolet detection. For the first time, a novel microwave assisted degradation procedure was employed for stress testing studies. In addition, orthogonal separation technique was applied to demonstrate selectivity of the proposed method. The method has demonstrated excellent linearity over the range of 25–1,600 ng mL^?1. Moreover, the method was found to be sensitive with a low limit of detection (3.35 ng mL^?1) and limit of quantitation (10.16 ng mL^?1). The method has shown good and consistent recoveries (99.35–100.69%) with low intra- and inter-day relative standard deviation (RSD) (<2.5%). Experimental design confirmed that peak area was unaffected by small changes in critical factors, in robustness study. The validated method was successfully applied for determination of IM in pharmaceutical formulations.     

A Stability Indicating LC Method for Amtolmetin Guacyl

A simple, sensitive gradient RP-LC assay method has been developed for the quantitative determination of amtolmetin guacyl in bulk drug, used as anti-inflammatory drug. The developed method is also applicable for related substances determination. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in a gradient mode and quantification was carried out using ultraviolet detection at 313 nm at a flow rate of 1.0 mL min^?1. In the developed LC method the resolution between Amtolmetin Guacyl and its three potential impurities was found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) greater than 0.99 for amtolmetin guacyl and its three impurities. This method was capable to detect all three impurities of amtolmetin guacyl at a level of 0.002% with respect to test concentration of 0.5 mg mL^?1 for a 10 μL injection volume. The inter- and intra-day precision values for all three impurities and for amtolmetin guacyl was found to be within 2.0% RSD at its specification level. The method has shown good and consistent recoveries for amtolmetin guacyl (99.2–101.5%) and its three impurities (94.5–104.8%). The test solution was found to be stable in diluent for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.6%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

Application of dispersive liquid–liquid microextraction for the determination of donepezil in urine by HPLC using a core–shell column

A rapid and novel method combining dispersive liquid–liquid microextraction and high-performance liquid chromatography coupled with fluorescence detection was developed for the determination of donepezil in human urine. Parameters affecting extraction efficiency and chromatographic determination, such as the type and volume of the extraction and disperser solvent, pH of sample for dispersive liquid–liquid microextraction, mobile-phase composition, pH, column oven temperature, and flow rate for chromatographic determination, were evaluated and optimized. Using a C₁₈ core–shell column (7.5 × 4.6?mm, 2.7?μm), the determination of donepezil was accomplished within 5?min. Under optimum conditions, developed method was linear in the range of 0.5–25?ng?mL^?1 with the correlation coefficient >0.99. Limit of detection was 0.15?ng?mL^?1. The relative standard deviation at three concentration levels (2, 12.5, and 20?ng?mL^?1) was less than 11% with accuracy in the range of 96.9–102.8%. The results of this study demonstrate that the use of dispersive liquid–liquid microextraction and core–shell column can be considered as a powerful tool for the analysis of donepezil in human urine.     

Development of the HPLC Method for Simultaneous Determination of Lidocaine Hydrochloride and Tribenoside Along with Their Impurities Supported by the QSRR Approach

A new liquid chromatographic (LC) method for simultaneous determination of lidocaine hydrochloride (LH) and tribenoside (TR) along with their related compounds in pharmaceutical preparations is described. Satisfactory LC separation of all analytes after the liquid–liquid extraction (LLE) procedure with ethanol was performed on a C₁₈ column using a gradient elution of a mixture of acetonitrile and 0.1 % orthophosphoric acid as the mobile phase. The procedure was validated according to the ICH guidelines. The limits of detection (LOD) and quantification (LOQ) were 4.36 and 13.21 μg mL^?1 for LH, 7.60 and 23.04 μg mL^?1 for TR, and below 0.11 and 0.33 μg mL^?1 for their impurities, respectively. Intra- and inter-day precision was below 1.97 %, whereas accuracy for all analytes ranged from 98.17 to 101.94 %. The proposed method was sensitive, robust, and specific allowing reliable simultaneous quantification of all mentioned compounds. Moreover, a comparative study of the RP-LC column classification based on the quantitative structure-retention relationships (QSRR) and column selectivity obtained in real pharmaceutical analysis was innovatively applied using factor analysis (FA). In the column performance test, the analysis of LH and TR in the presence of their impurities was carried out according to the developed method with the use of 12 RP-LC stationary phases previously tested under the QSRR conditions. The obtained results confirmed that the classes of the stationary phases selected in accordance with the QSRR models provided comparable separation for LH, TR, and their impurities. Hence, it was concluded that the proposed QSRR approach could be considered a supportive tool in the selection of the suitable column for the pharmaceutical analysis.     

Ion-Pair RP-LC of Tegaserod Maleate and its Impurities in Pharmaceutical Formulations and in Dissolution Studies

A simple ion-pair reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for determination of tegaserod maleate and related impurities in tablet dosage forms. The mobile phase was 60:40 (v/v) acetonitrile-25 mmol L^?1 sodium dodecyl sulfate, adjusted to pH 2.6 with glacial acetic acid. A C18 column was used as stationary phase and UV detection was at 314 nm. The method was optimized and validated. Response was linearly dependent on concentration between 0.1 and 100 µg mL^?1 with a limit of quantification (LOQ) of 0.1 µg mL^?1 for tegaserod maleate (S/N = 10). Under optimum conditions, tegaserod maleate was successfully separated from related substances, including 5-methoxyindole-3-carboxaldehyde remaining after synthesis and other impurities possibly resulting from oxidization and decomposition. The excipients did not interfere with assay of tegaserod maleate in tablet dosage forms. It is suggested that the proposed method can be used for routine quality control and dosage-form assay of tegaserod maleate.     

Stochastic resonance algorithm applied to quantitative analysis for weak liquid chromatographic signal of pyrene in water samples

The phenomenon of stochastic resonance (SR), which was discovered in recent years, rendered an entirely new way for the detection of weak signals, and it has been widely studied in many different science fields. This phenomenon is manifest in nonlinear systems whereby a weak signal can be amplified when the noise, signal and nonlinear system attain the proper cooperation. The introduced algorithm was employed to detect pyrene in drinking water samples with solid-phase extraction–liquid chromatography. The weak chromatographic peak of the analyte was amplified significantly, and the profiles of the peaks were also satisfactory. The limit of detection and the limit of quantification were improved from 0.022?ng?mL^?1 and 0.08?ng?mL^?1 to 0.004?ng?mL^?1 and 0.01?ng?mL^?1, respectively. The results showed an excellent quantitative relationship between concentrations and chromatographic responses. It is expected that the SR will be an effective tool to detect weak chromatographic peaks quantitatively in trace analysis.     

Quantification of Doxorubicin in Pegylated Polymer Micelles in Rat Plasma by Methanol Precipitation�CUltrasonic Emulsion Breaking Method and UPLC�CMS�CMS

This study established a new methanol precipitation?Cultrasonic emulsion breaking method for extraction of doxorubicin from polymeric micelles and developed a UPLC?CMS/MS method for the determination of doxorubicin in rat plasma. The emulsion breaking efficiency of methanol is up to 99.2%. The plasma samples were analyzed by UPLC/MS/MS using positive electrospray ionization mode in the multiple-reaction monitoring (MRM) mode. The calibration curves were linear over the range 5?C1,000 ng mL^?1 with the lower limit of quantification of 5 ng mL^?1. The intra- and inter-day precisions of QC samples were all <10.0%. The chromatographic separation was 2.5 min. The developed method was successfully applied to a pharmacokinetic study of doxorubicin in rats following intravenous administration.     

Development and validation of UPLC method for simultaneous quantification of carvedilol and ivabradine in the presence of degradation products using DoE concept

A methodical design-of-experiments were performed by applying quality-by-design concepts to establish a design-space for simultaneous and rapid quantification of Carvedilol and Ivabradine by UPLC in the presence of degradation products. Response-surface, central-composite design, and quadratic model were employed for statistical assessment of experimental data using the Design-Expert software. Response variables such as resolution and retention time were analyzed statistically for chromatographic screening. During DoE study, various plots such as perturbation, contour, 3D and design-space plots were considered for method optimization. The method was developed using C8 [100?×?2.1?mm, 1.8?µ] UPLC column, mobile phase comprising 0.5% triethylamine buffer [pH 6.4] and acetonitrile in the ratio of 50:50 v/v, the flow rate of 0.4?mL minute^?1 and UV detection at 285?nm for both Carvedilol and Ivabradine. The method was developed with a short run time of two minutes. The method was found to be linear in the range of 25.0–199.9?µg?mL^?1 and 8.9–21.3?µg?mL^?1 for Carvedilol and Ivabradine, respectively with a correlation coefficient of 0.9998 in each case. The recovery values were found in the range of 99.7–100.8% and 98.9–100.9% for Carvedilol and Ivabradine, respectively. The method was validated according to ICH Q2 (R1) guidelines.     

LC–MS–MS Simultaneous Determination of Paracetamol, Pseudoephedrine and Chlorpheniramine in Human Plasma: Application to a Pharmacokinetic Study

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed for the simultaneous determination of paracetamol, pseudoephedrine and chlorpheniramine in human plasma. Diphenhydramine was used as the internal standard. Analytes were extracted from alkalized human plasma by liquid–liquid extraction (LLE) using ethyl acetate. After electrospray ionization positive ion fragments were detected in the selected reaction monitoring (SRM) mode with a triple quadrupole tandem mass spectrometer. The method was linear in the concentration range of 20.0–10000.0 ng mL^?1 for paracetamol, 1.0–500.0 ng mL^?1 for pseudoephedrine and 0.1–50.0 ng mL^?1 for chlorpheniramine. The intra- and inter-day precisions were below 14.5% and the bias was between ?7.3 and +2.8% for all analytes. The validated LC–MS–MS method was applied to a pharmacokinetic study in which each healthy Chinese volunteer received a tablet containing 300 mg benorylate, 30 mg pseudoephedrine hydrochloride and 2 mg chlorpheniramine maleate. This is the first assay method described for the simultaneous determination of paracetamol, pseudoephedrine and chlorpheniramine in human plasma samples.     

Development and Validation of a New Analytical Method for the Determination of Related Components in Quetiapine Hemifumarate

A simple, sensitive isocratic rapid resolution liquid chromatographic assay method has been developed for the quantitative determination of quetiapine hemifumarate in bulk active pharmaceutical ingredient, used for the treatment of schizophrenia. The developed method is also applicable for the process related impurities determination. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in a isocratic mode and quantification was by ultraviolet detection at 225 nm at a flow rate of 1.0 mL min^?1. In the developed LC method the resolution between quetiapine hemifumarate and its three potential impurities was found to be greater than 2.0. Regression analysis showed an r value (correlation coefficient) greater than 0.99 for quetiapine hemifumarate and its three impurities. This method was capable to detect all three impurities of quetiapine hemifumarate at a level of 0.003% with respect to test concentration of 1.0 mg mL^?1 for a 3 μL injection volume. The bulk active pharmaceutical ingredient was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RR-LC method was validated with respect to linearity, accuracy, precision and robustness.     

Separation,Characterization, and Quantification of Atorvastatin and Related Impurities by Liquid Chromatography-Electrospray Ionization Mass Spectrometry

A method coupling high-performance liquid chromatography with diode-array detector and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MSⁿ) has been developed for the separation and characterization of atorvastatin and its related impurities. The results obtained using positive ion mode showed some diagnostic fragments that are useful for the identification of atorvastatin related impurities in real samples. Quantitative analysis of drug impurities was performed in the multiple reaction monitoring mode. Quantification limits for impurities were in the ranges 21.5–70.8 ng mL^?1. The method was successfully applied to the drug purity evaluation and quantitative determination of atorvastatin related impurities in bulk drugs and pharmaceutical formulations.     

Chemical modification of alumina surface by immobilization of 1-((5-nitrofuran-2-yl)methylene)thiosemicarbazide for extractive concentration of silver ions

A column, solid phase extraction (SPE), preconcentration method was developed for determination of silver by using alumina coated with 1-((5-nitrofuran-2-yl)methylene)thiosemicarbazide and determination by flame atomic absorption spectrometry. The separation/preconcentration conditions for the quantitative recovery were investigated. At pH 2, the maximum sorption capacity of Ag⁺ was 7.5?mg?g^?1. The linearity was maintained in the concentration range of 0.02–11.0?µg?mL^?1 in the final solution or 0.14–1.10?×?10^4?ng?mL^?1 in the original solution for silver. The preconcentration factor of 140 and relative standard deviation of ±1.4% was obtained, under optimum conditions. The limit of detection (LOD) was calculated as 0.112?ng?mL^?1, based on 3σ_bl/m (n?=?8) in the original solutions. The proposed method was successfully applied to the determination trace amounts of silver in the environmental samples such as tea, rice and wheat flour, mint, and real water samples.