Fluorescence Detection For Simultaneous Determination Research Articles

Application of MEKC and UPLC with Fluorescence Detection for Simultaneous Determination of Amlodipine Besylate and Bisoprolol Fumarate.

Two chromatographic methods were described for simultaneous determination of the antihypertensive drugs amlodipine besylate (AML) and bisoprolol fumarate (BIS). Method I applies micellar electrokinetic capillary chromatography using a deactivated fused silica capillary (25 cm effective length × 50 μm internal diameter). The background electrolyte consisted of 0.01 M borate buffer (pH 9.2) containing 0.025 M sodium dodecyl sulphate and methanol in the ratio of 80:20 (v/v). Valsartan (VAL) was used as an internal standard. Diode array detector was set at 238, 224, and 210 nm for measuring AML, BIS, and VAL, respectively. Method II involves using ultra-performance liquid chromatography with fluorescence detection. Zorbax SB-C8 column (2.1 × 100 mm, 1.8 μm particle size) was used with isocratic elution of the mobile phase composed of 0.1% trifluoroacetic acid, acetonitrile, and methanol in the ratio of 55:35:10 (v/v) at a flow rate of 0.6 mL/min. Fluorescence detection was done using excitation wavelengths 230 and 370 nm and emission wavelengths 305 and 450 nm for BIS and AML, respectively. Validation parameters were carefully studied including linearity, ranges, precision, accuracy, robustness, detection, and quantification limits. Method I showed good linearity over the range 10-100 μg/mL for both dugs. Method II's linear ranges were 0.001-0.1 and 0.02-1 µg/mL for BIS and AML, respectively. The proposed methods were successfully validated and applied for assay of the studied drugs in their fixed-dose combination tablets. To the best of our knowledge, this study suggests the first electro-chromatographic and LC with fluorescence detection methods for simultaneous determination of amlodipine and bisoprolol.

A fast and green reversed-phase HPLC method with fluorescence detection for simultaneous determination of amlodipine and celecoxib in their newly approved fixed-dose combination tablets.

A fast, green, sensitive, and accurate analytical method using high-performance liquid chromatography couple with fluorescence detection was established and validated for the simultaneous determination of amlodipine besylate and celecoxib in their recently approved fixed-dose combination tablets (1:20). Separation of the two drugs was achieved on C18 reversed-phase column (Thermo ODS Hypersil, 4.6 × 250mm, particle size 5µm) using acetonitrile:potassium phosphate buffer (50mM; pH 5.5, 60:40 v/v) as a mobile phase at 40°C, which eluted at a rate of 1mL/min. Detection was carried out with excitation and emission wavelengths of 360 and 446nm for amlodipine and 265 and 359nm for celecoxib, respectively. The method was linear over a concentration range of 0.05-2 and 0.05-10 µg/mL and limit of detection reached to 0.017 and 0.0167 µg/mL for amlodipine and celecoxib, respectively. The developed method was successfully applied to assess the cited drugs in their newly FDA approved fixed-dose combination tablet dosage form. Furthermore, the method was found to be sensitive and eco-friendly green alternative to the reported methods as it was evaluated according to the green analytical procedure index tool guidelines and analytical Eco-Scale.

A Rapid and Sensitive HPLC-FLD Method for the Determination of Retinol and Vitamin E Isomers in Human Serum

Background: Retinol and vitamin E are fat-soluble vitamins crucial for human health, yet their isomers’ distributions in the human body are still known roughly. In order to figure out the physical condition and evaluate the nutritional status of an individual, it is imperative to analyze retinol and VE isomers in human serum. Objective: This work aims to establish a rapid and simple high-performance liquid chromatography with fluorescence detection for simultaneous determination of retinol and vitamin E isomers in human serum. Methods: Separation was accomplished on a common C18 column thermostated at 25 oC, using a simple isocratic elution program of methanol/acetonitrile (85:15, v/v) at a flow rate of 1.0 mL/min. Fluorescence detection was operated using excitation/emission wavelengths of 329 nm/472 nm for retinol and 294 nm/338 nm for VE isomers, respectively. Results: Rapid separation was achieved within 13 min. Linear ranges of the method were 0.020-50.0 µg/mL, with correlation coefficients greater than 0.999. Detection limits and the quantification limits were 0.001-0.004 µg/mL and 0.003- 0.013 µg/mL, respectively. Mean recoveries were 84.1%- 98.2%, with intra-day and inter-day relative standard deviations less than 12.3% and 13.6%, respectively. This method has been applied to the simultaneous determination of retinol and 8 VE isomers in human serum samples with satisfactory results. Conclusion: A rapid, simple and robust method was developed for routine analysis of retinol and eight vitamin E isomers in human serum, providing a useful tool for clinical diagnosis and nutritional evaluation.

Development of HPLC method coupled with fluorescence detection for simultaneous determination of donepezil HCl and trazodone HCl in spiked human plasma and tablets dosage forms

Elderly people with dementia are commonly suffered from sleep disorders. So, the use of Donepezil hydrochloride as anti-Alzheimer drug and Trazodone hydrochloride as antidepressants with hypnotic action is very important in these cases. This study reports about novel and sensitive RP-HPLC method with fluorescence detection for simultaneous bioanalytical determination of Donepezil hydrochloride (DON) and co-administered, Trazodone hydrochloride (TRA) in their pure forms, spiked human plasma and tablets. Elution of both drugs was achieved with excellent resolution using a RP-C18 Hypersil Gold column and an isocratic mobile phase consisting of phosphate buffer (50mm, pH 4.6): methanol: acetonitrile (60:35:5) with a flow rate of 1.5mL/min and 20μL as injection volume. A Fluorescence detector at 300nm for excitation and 400nm for emission was used. Retention times were 4.3 and 6.3min for Donepezil hydrochloride and Trazodone hydrochloride, respectively. Linearity ranges of the assay were 25-1000 and 50-5000ng/mL and the limits of detection (LOD) and quantitation (LOQ) were 8.52, 15.47 and 25.81, 46.89ng/mL for Donepezil hydrochloride and Trazodone hydrochloride, respectively. The high sensitivity of the proposed method enabled the successful determination of the cited drugs in spiked human plasma with mean percentage of recoveries of 91.58±3.34 and 100.30±5.11 for Donepezil hydrochloride and Trazodone hydrochloride, respectively.

Simultaneous determination of 10 kinds of biogenic amines in rat plasma using high‐performance liquid chromatography coupled with fluorescence detection

We describe a simple, rapid, selective and sensitive HPLC method coupled with fluorescence detection for simultaneous determination of 10 kinds of biogenic amines (BAs: tryptamine, 2-phenethylamine, putrescine, cadaverine, histamine, 5-hydroxytryptamine, tyramine, spermidine, dopamine and spermine). BAs and IS were derivated with dansyl chloride. Fluorescence detection (λex /λem = 340/510 nm) was used. A satisfactory result for method validation was obtained. The assay was shown to be linear over the ranges 0.005-1.0 μg/mL for tryptamine, 2-phenethylamine and spermidine, 0.025-1.0 μg/mL for putrescine, 0.001-1.0 μg/mL for cadaverine, 0.25-20 μg/mL for histamine, 0.25-10 μg/mL for 5-hydroxytryptamine and dopamine, and 0.01-1.0 μg/mL for tyramine and spermine. The limits of detection and the limits of quantification were 0.3-75.0 ng/mL and 1.0-250.0 ng/mL, respectively. Relative standard deviations were ≤5.14% for intra-day and ≤6.58% for inter-day precision. The recoveries of BAs ranged from 79.11 to 114.26% after spiking standard solutions of BAs into a sample at three levels. Seven kinds of BAs were found in rat plasma, and the mean values of tryptamine, 2-phenethylamine, putrescine, cadaverine, histamine, spermidine and spermine determined were 52.72 ± 7.34, 11.45 ± 1.56, 162.56 ± 6.26, 312.75 ± 18.11, 1306.50 ± 116.16, 273.89 ± 26.41 and 41.51 ± 2.07 ng/mL, respectively.

A simple HPLC method with fluorescence detection for simultaneous determination of 10-methoxycamptothecin and its metabolite 10-hydroxycamptothecin in rat liver tissue.

A simple HPLC method to determine the amount of 10-methoxycamptothecin (MCPT) and its major metabolite 10-hydroxycamptothecin (HCPT) in rat liver tissue was developed in the present study. Camptothecin (CPT) was used as internal standard (IS). A piecewise linear function was used over lower and higher concentrations, respectively. The calibration curves were linear (r (2) >0.99) over concentrations from 2.5 to 20 ng/mL and 20 to 320 ng/mL for both MCPT and HCPT. The method had an accuracy of 92.74% to 112.76%, and the intra- and inter-day precision (RSD%) were 11.85% or less for MCPT and HCPT. The stability data showed no significant degradation occurred under the experimental conditions. This method was successfully applied to the tissue distribution study of MCPT and its metabolite HCPT in liver tissue samples after intravenous administration.

Development and validation of a RP-HPLC method with fluorescence detection for simultaneous determination of 10-methoxycamptothecin and its metabolite 10-hydroxycamptothecin in rat plasma

Both 10-methoxycamptothecin (MCPT) and 10-hydroxycamptothecin (HCPT) are the natural bioactive derivatives of camptothecin (CPT) isolated from Camptotheca acuminata, and have been confirmed to possess high anti-cancer properties. In the present study, HCPT was identified as the major metabolite of MCPT in rat plasma through HPLC/photodiode array detection (PDA) and LC–MS/MS analysis. A sensitive and reliable RP-HPLC method with fluorescence detection was developed and validated for the simultaneous analysis of MCPT and HCPT in rat plasma. The parental CPT was used as an internal standard (IS). A piecewise linear function was used over lower and higher concentrations, respectively. The calibration curves were linear (r2>0.999) over concentrations from 1.25 to 20ng/mL and 20 to 320ng/mL for both MCPT and HCPT. The method had an accuracy of 92.24–113.90%, and the intra- and inter-day precision (RSD%) were 10.05% or less for MCPT and HCPT. The stability data showed no significant degradation occurred under the experimental conditions. The mean recoveries at concentrations of 2.5, 40 and 160ng/mL were 95.09±3.94%, 98.67±1.40% and 95.65±2.15% for MCPT and 84.06±4.39%, 84.85±3.10% and 81.03±4.44% for HCPT, respectively. The lower limit of quantification (LLOQ) using 0.1mL of plasma was 1.25ng/mL for both MCPT and HCPT. This method was successfully applied to the pharmacokinetic study of MCPT and its metabolite HCPT in rat plasma after intravenous administration.

High‐performance liquid chromatographic determination of anti‐ hypertensive drugs on dried blood spots using a fluorescence detector – method development and validation

A selective and sensitive high-performance liquid chromatography method with fluorescence detection for simultaneous determination of irbesartan, losartan and valsartan on dried blood spots (DBS) has been developed and validated. It involves solvent extraction of a punch of DBS followed by reversed-phase liquid chromatography on a Lichrospher(®) 100 RP-18e column. Fluorescence detection was performed at 259 and 385 nm as excitation and emission wavelengths, respectively. The detection limits of irbesartan, losartan and valsartan were 1.8, 3.6 and 1.8 ng/mL respectively. The mean recoveries of irbesartan, losartan and valsartan were 98.68, 98.42 and 97.81%, respectively. The mean inter-day and intra-day precisions of irbesartan, losartan and valsartan were 2.07 and 1.34%, 1.42 and 1.48%, and 3.20 and 2.15% respectively. The proposed method was simple and rapid. Design of experiments was used to evaluate the robustness of the method.

Simultaneous determination of five anthraquinones in medicinal plants and pharmaceutical preparations by HPLC with fluorescence detection

A reversed-phase high-performance liquid chromatography (RP-HPLC) method with fluorescence detection for simultaneous determination of five anthraquinones in Rhubarb collected from nine different locations in China, Polygonum cuspidatum, Polygoni multiflori and three pharmaceutical preparations is proposed and validated. Chromatography was carried out at 25 °C on a Hypersil C18 column with the isocratic mobile phase of methanol–0.1% aqueous formic acid (85:15, v/v) at a flow rate of 1.0 ml/min. The excitation and emission wavelengths were set at 440 and 540 nm, respectively. A comprehensive validation of the method included tests of sensitivity, linearity, precision and accuracy. The linear regressions were acquired with r > 0.999. Satisfactory intra- and inter-day precisions were achieved with R.S.D.s less than 3.95% and the average recovery factors obtained were in the range of 93.2–103.8%.

Light-emitting diode-based indirect fluorescence detection for simultaneous determination of anions and cations in capillary electrophoresis

This report presents simultaneous analysis of cations and anions by capillary electrophoresis (CE) in conjunction with indirect fluorescence detection using a blue light-emitting diode (LED), based on the displacement of fluorescein with anionic EDTA–metal complexes and anions. A new focusing system combined with a plastic lens and a 40× objective was developed and used effectively to focus the diverging beam of the LED on the capillary. The optimum compositions for simultaneous analysis of metal ions and anions are the samples prepared in 5 mM borate, pH 9.2, containing 2 mM EDTA and the background electrolytes (BGEs) consisting of 5 mM borate buffer, 5 μM fluorescein, and 1 μM NaCl at pH 9.2. Using this pre-capillary complexation method, the analysis of a sample containing five metal ions and eight anions was accomplished in 8 min, with the relative standard deviation values for the migration times less than 2.0%. The peak heights against the concentrations of the metal ions and anions are linear in 10–1000 and 50–2000 μM, with correlation coefficients better than 0.998, and 0.982, respectively. The limits of detection at a signal-to-noise ratio 3 of up to 14.6 μM for formate and as low as 3.7 μM for Ni 2+. The results of the analyses of pond water and a Chinese herbal soup present the advantages of this method, including simplicity, rapidity, reproducibility, and low costs.

Quantification of imipramine and its major metabolites in whole blood, brain, and other tissues of the rat by liquid chromatography.

An assay procedure is presented using high-performance liquid chromatography and fluorescence detection for simultaneous determination of imipramine, desipramine, and their 2-hydroxylated metabolites in whole blood and various tissues of the rat. By modifying methods previously described for plasma, we have developed an assay method for sensitive, reproducible quantitation of these compounds in rat plasma, whole blood, brain, liver, and fetus. Increasing the volume of the extraction solvent (20% butanol in hexane) increased recovery of imipramine and desipramine to greater than 90% for all tissues examined. No interfering peaks were observed for any of the tissues studied. Addition of n-butylamine as a modifier to the mobile phase decreased the retention of all four compounds and decreased peak tailing of the demethylated compounds. The total elution time was less than 15 min. Using these methods the coefficient of variation for all four compounds was generally less than 5% for each of the five tissues examined. The calibration curves were linear for standard concentrations of 25-2,000 ng/ml spiked tissue homogenate. This method has sufficient sensitivity and specificity for use in pharmacokinetic studies of imipramine.