Simultaneous Determination and Drug Dissolution Testing of Combined Amlodipine Tablet Formulations Using RP-LC

In the proposed work, the simultaneous analysis of amlodipine–rosuvastatin and amlodipine–atorvastatin in their dosage forms was achieved. Simultaneous dissolution profiles of the amlodipine–rosuvastatin and amlodipine–atorvastatin tablets are realized using Apparatus II with a simple, accurate and precise RP-LC method. The mobile phase consisting of 0.2 % H₃PO₄ and pH 5:methanol:acetonitrile (46:27:27) was used. The samples of 10 µL were injected onto a Zorbax SB C18 (100 mm, 4.6 mm, 3.5 µm particle size) column with 1.2 µL min⁻¹ flow rate. The samples were detected at 236 nm. By plotting peak area ratios vs. concentration, the linearity for amlodipine–rosuvastatin and amlodipine–atorvastatin was determined. With the developed RP-LC method, AML, ROS and ATOR were detected within the range of 0.25–10, 0.5–10 and 0.25–25 µg mL⁻¹, respectively. LOD and LOQ values were also calculated as 0.028, 0.058, 0.021 and 0.095 µg mL⁻¹, 0.195 µg mL⁻¹, 0.070 µg mL⁻¹ for AML, ROS and ATOR, respectively. System suitability tests parameters, such as capacity factor, selectivity to previous peak, selectivity to next peak, resolution to previous peak, resolution to next peak, tailing factor, theoretical number of plates, were performed and found coherent with the ICH guideline parameters. The proposed method has been extensively validated in terms of recovery, and recovery results were between 99 and 101 %. For proving the precision, between-day and within-day repeatability results of the method were proposed. The method can be used for the simultaneous determination of amlodipine–rosuvastatin and amlodipine–atorvastatin.

     

Rapid and Highly Sensitive HPLC and TLC Methods for Quantitation of Amlodipine Besilate and Valsartan in Bulk Powder and in Pharmaceutical Dosage Forms and in Human Plasma

Two simple, sensitive, and specific high-performance liquid chromatography and thin-layer chromatography methods were developed for the simultaneous estimation of Amlodipine besilate (AM) and Valsartan (VL). Separation by HPLC was achieved using a xTerra C18 column and methanol /acetonitrile /water/ 0.05% triethylamine in a ratio 40:20:30:10 by volume as mobile phase, pH was adjusted to 3 ± 0.1 with o-phosphoric acid. The flow rate was 1.2 mL min^?1. The linearity range was 0.2 to 2 µg mL^?1 for amlodipine besilate and 0.4 to 4 µg mL^?1 for Valsartan with a mean percentage recovery of 99.59 ± 0.523% and 100.61 ± 0.400% for amlodipine besilate and valsartan, respectively. The TLC method used silica gel 60 F₂₅₄ plates; the optimized mobile phase was ethyl acetate/ methanol / ammonium hydroxide (55:45:5 by volume). Quantitatively, the spots were scanned densitometrically at 237 nm. The range was 0.5–4.0 µg spot^?1 for amlodipine besilate and 2.0–12.0 µg spot^?1 for valsartan. The mean percentages recovery was 99.80 ± 0.451% and 100.61 ± 0.363% for amlodipine besilate and valsartan, respectively. The HPLC method was found to be simple, selective, precise, and reproducible for the estimation of both drugs from spiked human plasma.     

CE, with Hydroxypropyl-β-Cyclodextrin as Chiral Selector, for Separation and Determination of the Enantiomers of Amlodipine in the Serum of Hypertension Patients

A capillary electrophoretic method for separation of the enantiomers of amlodipine in the serum of hypertension patients has been established and validated. The two enantiomers were separated in a fused-silica capillary with phosphate running buffer (75 mmol L^?1, pH 2.5) containing 15 mmol L^?1 hydroxypropyl-β-cyclodextrin (HP-β-CD). The effects on the separation of buffer pH and concentration, separation potential, and concentration of HP-β-CD were investigated. The range of quantitation for both enantiomers was 2.0–16.0 μg mL^?1. Intra-day and inter-day relative standard deviation (RSD; n = 5) was <10%. The limits of detection (LOD) and quantification (LOQ) of the amlodipine enantiomers, at 214 nm, were approximately 0.5 and 0.7 μg mL^?1, respectively (S/N = 3 and 10, respectively; 5-s injection). Recovery was always >85%. Results from enantiomer separation and quantification showed that concentrations of the enantiomers of amlodipine in serum from an elderly patient were higher than in serum from a young patient administered the same dose. The method was useful for determining the concentration of the enantiomers of amlodipine in hypertension patient serum and for monitoring the transition behavior of the enantiomers in humans. The method proved suitable for application to the separation of the enantiomers of amlodipine and analysis of clinical samples.     

HPLC�CMS�CMS for the Simultaneous Determination of Atorvastatin and Amlodipine in Plasma of Hypertensive Patients

Combination drug products containing amlodipine and atorvastatin are widely marketed and used in the treatment of concomitant hypertension and dyslipidemia. A rapid, simple and sensitive high performance liquid chromatography?Ctandem mass spectrometry (HPLC?CMS?CMS) method for determination of atorvastatin and amlodipine in plasma of hypertensive patients has been developed and validated to be used for therapeutic drug monitoring. The plasma samples were subjected to methanol protein precipitation. Chromatographic separation was performed on a C18 column using a gradient elution. The mobile phase consisted of 0.1% of formic acid in water and 0.1% of formic acid in acetonitrile and was pumped at a flow rate of 0.4 mL min^?1. Detection of analytes was achieved by tandem mass spectrometry with electrospray ionization (ESI) interface in positive ion mode. The calibration curves were linear over the range of 0.46?C1,000 ng mL^?1. The intra- and inter-day precisions were within 12.2%, while the accuracy ranged from 92.7 to 108.1%. The validated LC?CMS?CMS method was successfully applied for the determination of atorvastatin and amlodipine in plasma of hypertensive patients.     

99mTc-nebivolol as a novel heart imaging radiopharmaceutical for myocardial infarction assessment

Non-invasive quantification of myocardial β-receptors could become an independent prognostic marker for chronic heart failure and cardiovascular disorders. The aim of this study was to formulate a novel radiopharmaceutical for the detection of myocardial infarction at early stages in susceptible patients, which requires the development of high myocardium affinity radiopharmaceuticals able to establish an accurate in vivo quantification of cardiac β₁-adrenoceptors. This was attained by the direct complexation of nebivolol as a cardioselective agent with technetium-99m as one of the most useful radionuclides in diagnostic nuclear medicine. Factors affecting the radiochemical yield such as nebivolol amount, stannous chloride amount, reaction time and pH of the reaction mixture were optimized. The results showed that the radiochemical yield was 95 ± 2.87 % and the radiolabeled compound was separated by high performance liquid chromatography. In vitro studies showed that the formed complex was stable for up to 24 h. In vivo uptake of ^99mTc-nebivolol in the heart was 4.55 ± 0.23 % ID/g organ at 0.5 h post injection, whereas the clearance from Albino mice appeared to proceed via the hepatobiliary and renal clearance pathways. Predosing mice with cold nebivolol reduced the heart uptake to 1.1 ± 0.02 % and further confirmed the high specificity and selectivity of this radiotracer for the assessment of the myocardial β₁-adrenoceptors.     

Robust UHPLC Separation Method Development for Multi-API Product Containing Amlodipine and Bisoprolol: The Impact of Column Selection

This paper describes a new and fast ultra-high pressure liquid chromatographic separation of amlodipine and bisoprolol and all their closely related compounds, for impurity profiling purposes. Computer-assisted method development was applied and the impact of several state-of-the-art stationary phase column chemistries (50 × 2.1 mm, sub-2 μm, and core–shell type materials) on the achievable selectivity and resolution was investigated. The work was performed according to quality by design principles using design of experiment with three experimental factors; namely the gradient time (t _G), temperature (T), and mobile phase pH. Thanks to modeling software, it was proved that the separation of all compounds was feasible on numerous column chemistries within <10 min, by proper adjustments of variables. It was also demonstrated that the reliability of predictions was good, as the predicted retention times and resolutions were in good agreement with the experimental ones. The final, optimized method separates 16 peaks related to amlodipine and bisoprolol within 7 min, ensuring baseline resolution between all peak-pairs.     

Stability-indicating method for the determination of assay and quantification of impurities in amlodipine–atorvastatin combination dosage form by RP-HPLC

A simple stability-indicating RP-HPLC method was developed and validated for quantification of amlodipine, atorvastatin, and its impurities on Waters HPLC using Unisol C₁₈ 5?µm, 250?×?4.6?mm column in their combined tablet dosage as per ICH guidelines. The gradient (T/%B) at 0/42, 18/42, 22/75, 30/75, 32/42, and 35/42 of 40?mM 4.7 pH ammonium acetate as mobile phase A and acetonitrile as mobile phase B of flow rate 1.5?mL/min and 240?nm wavelength. Peak purity compiled for amlodipine and atorvastatin in all stressed conditions. For impurities: Precision was found in between 1.5 and 3.6%. The limit of detection and quantification for amlodipine, amlodipine impurity A, and atorvastatin was found to be 0.06 and 0.18?µg/mL, for atorvastatin Impurity A, B, C, and H was determined as 0.04 and 0.11?µg/mL, for Atorvastatin Impurity D was measured as 0.11 and 0.28?µg/mL, respectively. The linear regression achieved >0.9999 from 0.22 to 7.5?µg/mL. Recovery was observed in between 97 and 101%. For assay: Precision was determined in between 0.1 and 0.2%. The linear regression achieved >0.9999 for amlodipine and atorvastatin. Recovery ranged from 100 to 101%. The validated method was found to be accurate, precise, reliable, and robust to determine the assay as well as impurities in amlodipine–atorvastatin combination dosage formulation.     

Robust UHPLC Separation Method Development for Multi-API Product Containing Amlodipine and Bisoprolol: The Impact of Column Selection

This paper describes a new and fast ultra-high pressure liquid chromatographic separation of amlodipine and bisoprolol and all their closely related compounds, for impurity profiling purposes. Computer-assisted method development was applied and the impact of several state-of-the-art stationary phase column chemistries (50 × 2.1 mm, sub-2 μm, and core–shell type materials) on the achievable selectivity and resolution was investigated. The work was performed according to quality by design principles using design of experiment with three experimental factors; namely the gradient time (t _G), temperature (T), and mobile phase pH. Thanks to modeling software, it was proved that the separation of all compounds was feasible on numerous column chemistries within <10 min, by proper adjustments of variables. It was also demonstrated that the reliability of predictions was good, as the predicted retention times and resolutions were in good agreement with the experimental ones. The final, optimized method separates 16 peaks related to amlodipine and bisoprolol within 7 min, ensuring baseline resolution between all peak-pairs.

     

A Validated RP–LC Method for Simultaneous Determination of Losartan Potassium and Amlodipine Besilate in Pharmaceutical Preparations

A simple RP–LC method for simultaneous quantification of losartan and amlodipine and separation of their degradation products has been developed. For this purpose we tested appropriated mobile phase pH range, flow rate, temperature and different columns. The method was validated with an ODS column. A gradient of acetonitrile and phosphate pH 3.0 buffer was utilized as mobile phase. The linearity was determined at 50–150% level. Individual recoveries at 70–130% level ranged from 98.8 to 100.5% for losartan and 96.4–101.2% for amlodipine. The robustness was also evaluated. Although losartan has much higher quantities than amlodipine in commercial tablets, this method allowed simultaneous quantification for both drugs.     

Development of a Monoclonal Antibody-Based ELISA for the Detection of Oxytetracycline and 4-Epi-Oxytetracycline Residues in Chicken Tissues

In this study, a specific monoclonal antibody (Mab) against oxytetracycline (OTC) and its metabolite 4-epi-Oxytetracycline (4-epi-OTC) was produced. Based on this Mab, a sensitive and reliable method indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the detection of OTC and 4-epi-OTC from chicken meats. The ic-ELISA showed a 50% inhibition (IC₅₀) value of 2.01 ± 0.16 ng/ml and a detection limit of 0.13 ± 0.03 ng/ml. The recoveries from chicken muscles and livers spiked with OTC of 50–600 ng/g were 83.33–88.25% and 84.62–86.12%, respectively. The intra-assay coefficients of variation (CVs) were 4.73–9.31%, and the inter-assay CVs were 6.44–11.01%. The method showed a positive correlation with the traditional method HPLC (R² = 0.997) within a certain concentration of OTC used in this assay. The method developed in this study was simple and independent of specific expensive equipment. Thus, it could be useful as a convenient method to detect OTC residues.     

Residue Analysis of Propionylbrassinolide in Fruit and Vegetables by GC–MS

A rapid and simple method for the determination of propionylbrassinolide residues in tomatoes, apples and grapes using GC–MS is reported. Samples were extracted with acetonitrile, and the extracts were analyzed without any further clean-up. The results showed good linearity (r ² > 0.99) with standard solutions over the concentration range of 0.5–50 mg L^?1. The LODs and LOQs of propionylbrassinolide were 0.15 and 0.5 mg kg^?1 in all samples. Recoveries were in the range of 81.9–111.2%, with corresponding RSDs of 4.6–12.9% for three fortified levels. Intra- and inter-day RSDs were in the ranges of 1.5–14.2% and 5.3–15.6%. It was demonstrated that the proposed method is simple and efficient, and particularly suitable for detecting propionylbrassinolide residues in fruit and vegetables.     

Highly Sensitive LC Method with Automated Co-Sense System and Fluorescence Detection for Determination of Sertraline in Human Plasma

A highly sensitive LC method with column-switching “Co-sense” system and fluorescence detection has been proposed for trace determination of sertraline in human plasma. A simple pre-column derivatization procedure with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reagent was employed. Fluxetine was used as an internal standard. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9997) was found between the peak area ratio and sertraline concentration in the range of 5–5,000 ng mL^?1. The limit of detection and limit of quantitation were 1.41 and 4.28 ng mL^?1, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 5.63%. The accuracy of the method was proved; the recovery of sertraline from the spiked human plasma was 99.76–102.62 ± 2.19–5.63%. The proposed method had high throughput as the analysis involved simple sample pre-treatment procedure and short run-time (~12 min). The results demonstrated that the method would have a great value if applied in bioavailability and pharmacokinetic studies for sertraline.     

Rapid LC-UV Analysis of Iohexol in Canine Plasma for Glomerular Filtration Rate Determination

A rapid high-performance liquid chromatography UV method and a simple sample preparation for analyzing iohexol in canine plasma, for evaluating glomerular filtration rate (GFR) and intestinal permeability, were developed and validated. Trifluoroacetic acid (TFA) was used for protein precipitation and iohexol extraction from plasma, followed by vortex mixing and centrifugation. As an internal standard, 4-aminobenzoic acid (para-aminobenzoic acid, PABA) was added. The supernatant (5 μL) was injected into a Zorbax SB-C18 LC column maintained at 50 °C. The mobile phase of the LC method was a water–methanol gradient at pH 3.0 adjusted with TFA. Fast LC measurement was achieved by using a rapid-resolution LC technique. Total run time was 13 min, and UV wavelength was set at 246 nm. Precision of the method was 0.2–9.0%, depending on the iohexol concentration in plasma. Recovery of iohexol from plasma was over 90%, and recovery of the internal standard 99.1 ± 1.4%. The calibration curve was linear (r = 0.9997) over iohexol concentrations of 2.5–150 μg mL^?1 (n = 5). This method is fast, simple, reliable and applicable in clinical settings.     

Residue Analysis of Propionylbrassinolide in Fruit and Vegetables by GC–MS

A rapid and simple method for the determination of propionylbrassinolide residues in tomatoes, apples and grapes using GC–MS is reported. Samples were extracted with acetonitrile, and the extracts were analyzed without any further clean-up. The results showed good linearity (r ² > 0.99) with standard solutions over the concentration range of 0.5–50 mg L⁻¹. The LODs and LOQs of propionylbrassinolide were 0.15 and 0.5 mg kg⁻¹ in all samples. Recoveries were in the range of 81.9–111.2%, with corresponding RSDs of 4.6–12.9% for three fortified levels. Intra- and inter-day RSDs were in the ranges of 1.5–14.2% and 5.3–15.6%. It was demonstrated that the proposed method is simple and efficient, and particularly suitable for detecting propionylbrassinolide residues in fruit and vegetables.

     

Determination of Flavonoids in Costus speciosus and Etlingera elatior by Liquid Chromatography-Mass Spectrometry

In this article, a new and relatively simple liquid chromatography-mass spectrometry (LC-MS) method was developed for the simultaneous determination of five flavonoid compounds. Under optimized conditions, the LC-MS analysis can be achieved on a reverse-phase C₁₈ column (50 × 2.1 mm, 1.8 µm), with a binary mobile phase consisting of 0.1% formic acid in water and methanol under gradient elution conditions. The relative standard deviations (RSDs) of the retention times and peak areas for all analytes were in the range of 0.1–0.5% and 3.2–5.2%, respectively. The LODs and LOQs were in the range of 2.1–7.0 ng mL^?1 and 6.9–23 ng mL^?1, respectively. Furthermore, the developed LC-MS method was successfully applied to analyze flavonoids in different parts of two medicinal Zingiberaceae plants (i.e., Costus speciosus and Etlingera elatior) from different sources. The proposed LC-MS method was simple, effective, and reliable, and thus it has potential to be used for the quality control of other medicinal herbs.     

Selective LC Determination of Cabergoline in the Bulk Drug and in Tablets: In Vitro Dissolution Studies

Cabergoline (CAB) is an ergot alkaloid derivative with dopamine agonist activity. A novel, simple, and rapid stability-indicating high-performance liquid chromatographic (HPLC) method for assay of CAB in tablets has been developed and validated. Chromatography was performed on a 4.6 mm i.d. × 250 mm, 5 μm particle, cyano column with acetonitrile–10 mM phosphoric acid, 35:65 (v/v), containing 0.04% triethylamine, as mobile phase, at a flow rate of 1.0 mL min^?1, and UV detection at 280 nm. Response was a linear function of concentration in the range 0.1–4 μg mL^?1 (r ² = 0.9999). The recovery of the method was good (99.45%) and RSD values for intra-day and inter-day precision were 0.24–0.88% and 0.66–1.19%, respectively. The method can be used for quality-control assay of CAB in tablets, for stability studies, and for in vitro dissolution studies.     

Ion-Pair LC Method for Simultaneous Determination of Aliskiren Hemifumarate,Amlodipine Besylate and Hydrochlorothiazide in Pharmaceuticals

A rapid and precise LC method was developed for the simultaneous determination of aliskiren hemifumarate (ALS), amlodipine besylate (AML) and hydrochlorothiazide (HCZ) using acetonitrile:25 mM octane sulfonic acid sodium salt monohydrate in water (60:40 v/v) as the mobile phase. The flow rate was maintained at 1.2 mL min^?1 on a stationary phase composed of Supelco, Discovery^® HS (C18) column (25 cm × 4.6 mm, 5 μm). Isocratic elution was applied throughout the analysis. Detection was carried out at λ _max (232 nm) at ambient temperature. The method was validated according to ICH guidelines. Linearity, accuracy and precision were satisfactory over the concentration ranges of 32–320, 2–44 and 4–64 μg mL^?1 for ALS, AML and HCZ, respectively. LOD and LOQ were estimated and found to be 0.855 and 2.951 μg mL^?1, respectively, for ALS, 0.061 and 0.202 μg mL^?1, respectively, for AML as well as 0.052 and 0.174 μg mL^?1, respectively, for HCZ. The method was successfully applied for the determination of the three drugs in their co-formulated tablets. The results were compared statistically with reference methods and no significant difference was found. The developed method is specific and accurate for the quality control and routine analysis of the cited drugs in pharmaceutical preparations.     

Development of a Time-Resolved Fluoroimmunoassay for the Rapid Detection of Methyl-3-Quinoxaline-2-Carboxylic Acid in Porcine Tissues

A time-resolved fluoroimmunoassay for the specific determination of methyl-3-quinoxaline-2-carboxylic acid in animal tissues, a marker residue of olaquindox, was developed. The IC₅₀ of the assay was found to be 1.46 ± 0.19 ng/mL of methyl-3-quinoxaline-2-carboxylic acid in phosphate-buffered saline samples and the detection limit was 0.16 ± 0.03 ng/mL. For porcine liver and muscle samples spiked with 5, 10, and 15 ng/g, the recovery ranges were 95.7–112.3% and 98.5–116.2% and the coefficients of variation were 9.3–11.5% and 8.9–14.2%, respectively. The time-resolved fluoroimmunoassay results correlated well with high performance liquid chromatography results (correlation coefficients of 0.991 for liver and 0.988 for muscle). This study suggests that this method is simple, fast, and sensitive for the high-throughput determination of methyl-3-quinoxaline-2-carboxylic acid in animal tissues.     

A Simple Stability Indicating CZE Method for the Analysis of Octreotide Acetate

A simple stability indicating capillary zone electrophoretic method was developed and validated for the analysis of octreotide acetate (OCT-Ac). The best separation was achieved by bare fused silica capillaries (50 μm i.d.; 65.5 cm total and 57.0 cm effective length), phosphate buffer (pH = 3.25; 50 mM), at 32.5 °C. The samples were injected using 50 mbar for 5 s and subjected to the applied voltage of 27.5 kV for separation. The detection was carried out using a PAD at a wavelength of 195 nm. For improving the repeatability of the method, l-histidine was applied as an internal standard. According to the validation results, the method was linear in the concentration range of 3.30–400 μg mL^?1 (correlation coefficient of 0.9996) with a limit of detection of 1.08 μg mL^?1 and a limit of quantification of 3.30 μg mL^?1; accuracy of the method was between 100.4 ± 0.2 and 101.1 ± 0.2 %; intra-assay precision was 0.5–2.6 % and intermediate precision was 1.3–3.2 %. The proposed method was successfully applied for the quantification of OCT-Ac in both a pharmaceutical formulation and force-degraded samples and for the detection and separation of degradation products; besides, the obtained results were used for the evaluation of the degradation kinetics of OCT-Ac under different stress conditions. So, it is concluded that the developed method could be employed as a simple, accurate and precise stability-indicating method in quality control laboratories to assess the quantity and stability of OCT-Ac pharmaceutical products.     

Sensitive LC–ESI–MS Method for Determination of Tiopronin in Human Plasma

A sensitive liquid chromatographic–electrospray ionization–mass spectrometric (LC–MS) method has been developed for direct measurement of the concentration of tiopronin in human plasma. Hydrochloric acid solution was used to stabilize the tiopronin and prevent formation of a dimer, or reaction with endogenous thiols. The method involved liquid–liquid extraction of tiopronin from plasma samples with ethyl acetate, simple reversed-phase chromatography, and mass spectrometric detection with nanogram detection limits. Acetaminophen was used as internal standard (IS). The limit of quantification was 5 ng mL^?1 (RSD 4.3%). The method was validated within the linear range 5–500 ng mL^?1. The correlation coefficient for the calibration regression line was 0.9997 or better. Intra-day and inter-day accuracy were better than 15%. The method has been successfully used for a pharmacokinetic study with human subjects. Among the pharmacokinetic data obtained, t _1/2 was 2.37 ± 0.63 h and T _max was 4 h.