Development and Validation of a New Method for the Determination of Anti-hepatitis C Agent Simeprevir in Human Plasma using HPLC with Fluorescence Detection

2020 ◽  
Vol 16 (4) ◽  
pp. 428-435
Author(s):  
Ahmed F.A. Youssef ◽  
Yousry M. Issa ◽  
Kareem M. Nabil
Keyword(s):  
Hepatitis C ◽  
Human Plasma ◽  
Fluorescence Detection ◽  
Internal Standard ◽  
Protein Precipitation ◽  
Assay Method ◽  
Fluorescence Detector ◽  
Limit Of Quantification ◽  
Relative Standard

Background: Simeprevir is one of the recently discovered drugs for treating hepatitis C which is one of the major diseases across the globe. Objective: The present study involves the development of a new and unique High-Performance Liquid Chromatography (HPLC) method using fluorescence detection for the determination of simeprevir (SIM) in human plasma. Methods: Two methods of extractions were tested, protein precipitation using acetonitrile and liquidliquid extraction. A 25 mM dipotassium hydrogen orthophosphate (pH 7.0)/ACN (50/50; v/v), was used as mobile phase and C18 reversed phase column as the stationary phase. The chromatographic conditions were optimized and the concentration of simeprevir was determined by using the fluorescence detector. Cyclobenzaprine was used as an internal standard. Results: Recovery of the assay method based on protein precipitation was up to 100%. Intra-day and inter-day accuracies range from 92.30 to 107.80%, with Relative Standard Deviation (RSD) range 1.65-8.02%. The present method was successfully applied to a pharmacokinetic study where SIM was administered as a single dose of 150 mg SIM/capsule (Olysio®) to healthy individuals. Conclusion: This method exhibits high sensitivity with a low limit of quantification 10 ng mL-1, good selectivity using fluorescence detection, wide linear application range 10-3000 ng mL-1, good recovery and highly precise and validation results. The developed method can be applied in routine analysis for real samples.

scholarly journals Nonextractive Procedure and Precolumn Derivatization with 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole for Trace Determination of Trimetazidine in Plasma by High-Performance Liquid Chromatography with Fluorescence Detection

2008 ◽  
Vol 91 (5) ◽  
pp. 1037-1044 ◽  
Author(s):  
Ibrahim A Darwish ◽  
Ashraf M Mahmoud ◽  
Nasr Y Khalil
Keyword(s):  
Human Plasma ◽  
Fluorescence Detection ◽  
High Performance ◽  
Internal Standard ◽  
High Performance Liquid Chromatographic ◽  
Pharmacokinetic Studies ◽  
Precolumn Derivatization ◽  
Trace Determination ◽  
Relative Standard

Abstract A highly sensitive high-performance liquid chromatographic method with fluorescence detection has been developed and validated in a single laboratory for the trace determination of trimetazidine (TMZ) in human plasma. Fluoxetine (FLX) was used as the internal standard. TMZ and FLX were isolated from plasma by protein precipitation with acetonitrile and derivatized by heating with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole in pH 8 borate buffer at 70C for 30 min. Separations were performed in the isocratic mode on a Nucleosil CN column with the mobile phase acetonitrile10 mM sodium acetate buffer (pH 3.5)methanol (47 + 47 + 6, v/v/v) at a flow rate of 1.0 mL/min. The derivatized samples were excited at 470 nm and monitored at an emission wavelength of 530 nm. Under the optimum chromatographic conditions, a linear relationship with a good correlation coefficient (r 0.9997, n 5) was obtained for the peak area ratio of TMZ to FLX and for TMZ concentrations of 1120 ng/mL. The proposed method has the lowest limits of detection and quantitation reported to date for the determination of TMZ in plasma with values of 0.3 and 0.95 ng/mL, respectively. The values for intra- and interassay precision were satisfactory; the relative standard deviations were 4.04. The accuracy of the method was demonstrated; the recoveries of TMZ from spiked human plasma were 98.13102.83 0.24.04. The method has high throughput because of its simple sample preparation procedure and short run time (<10 min). The results demonstrated that the proposed method would have great value when applied in pharmacokinetic studies for TMZ.

scholarly journals Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with fluorescence detection

2011 ◽  
Vol 61 (4) ◽  
pp. 403-413 ◽  
Author(s):  
Mohammed Abonassif ◽  
Mohammed Hefnawy ◽  
Mohamed Kassem ◽  
Gamal Mostafa
Keyword(s):  
Human Plasma ◽  
Fluorescence Detection ◽  
High Performance ◽  
Internal Standard ◽  
Pharmaceutical Formulations ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Relative Standard ◽  
Donepezil Hydrochloride

Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with fluorescence detectionA sensitive, isocratic reversed-phase high performance liquid chromatographic method involving fluorescence detection was developed for the determination of donepezil hydrochloride in tablets and in human plasma. Pindolol was used as an internal standard. Good chromatographic separation was achieved by using an analytical column C18. The system operated at room temperature using a mobile phase consisting of methanol, phosphate buffer (0.02 mol L-1) and triethyl amine (pH 3.5) (55: 45: 0.5,V/V/V) at a flow rate 0.9 mL-1min. The analyte and internal standard were extracted from human plasmavialiquid-liquid extraction. The proposed method was validated for sensitivity, selectivity, linearity, accuracy and precision. The calibration curve was linear over the range of 5-2000 ng mL-1of donepezil with detection limit of 1.5 ng mL-1. Intra- and inter-day relative standard deviations were less than 2.5 %. The method was found to be suitable for quality control of donepezil hydrochloride in bulk drug as well as in human plasma.

scholarly journals Quantitative Determination of Azithromycin in Human Plasma by Liquid Chromatography–Mass Spectrometry and its Application in Pharmackokinetic Study

2012 ◽  
Vol 11 (1) ◽  
pp. 55-63 ◽  
Author(s):  
Maizbha Uddin Ahmed ◽  
Mohammad Safiqul Islam ◽  
Tasmin Ara Sultana ◽  
AGM Mostofa ◽  
Muhammad Shahdaat Bin Sayeed ◽  
...  
Keyword(s):  
Mobile Phase ◽  
Solid Phase ◽  
Internal Standard ◽  
Extraction Process ◽  
Serum Samples ◽  
Limit Of Quantification ◽  
Relative Standard ◽  
Linear Concentration Range ◽  
Linear Concentration

Azithromycin is an effective and well-known antimicrobial agent. In the present study, a simple, sensitive and specific LC/MS/MS method has been developed and validated for the quantification of Azithromycin in  human serum samples using Clarithromycin as internal standard. Azithromycin was extracted from biological matrix  by using solid phase extraction process. The chromatographic separation was performed on Luna C18 (3 ?, 2x150   mm) column with a mobile phase consisting of 35 mM ammonium acetate buffer (mobile phase-A) and acetonitrile  and methanol in ratio of 90:10 ( as mobile phase-B) at a flow rate of 0.25 mL/min. The method was validated over a  linear concentration range of 0.5?50.0 ng/mL and limit of quantification (LOQ) was 0.5 ng/mL with a coefficient of  correlation (r2) = 0.9998. The intra-day and inter-day precision expressed as relative standard deviation were 1.64% – 8.43% and 2.32% – 9.92%, respectively. The average recovery of azithromycin from serum was 98.11%. The method  was successfully applied to a pharmacokinetic study after oral administration of Azithromycin 200 mg/5 ml suspension in healthy Bangladeshi volunteers. DOI: http://dx.doi.org/10.3329/dujps.v11i1.12488 Dhaka Univ. J. Pharm. Sci. 11(1): 55-63, 2012 (June)

scholarly journals A Modified Method for Determination of Lumefantrine in Human Plasma by HPLC-UV and Combination of Protein Precipitation and Solid-Phase Extraction: Application to a Pharmacokinetic Study

2010 ◽  
Vol 5 ◽  
pp. ACI.S4431 ◽  
Author(s):  
Liusheng Huang ◽  
Patricia S. Lizak ◽  
Anura L. Jayewardene ◽  
Florence Marzan ◽  
Ming-Na Tina Lee ◽  
...  
Keyword(s):  
Solid Phase Extraction ◽  
Human Plasma ◽  
Solid Phase ◽  
Internal Standard ◽  
Pharmacokinetic Interaction ◽  
Gradient Elution ◽  
Protein Precipitation ◽  
Phase Extraction ◽  
Modified Method

An HPLC-UV method was developed and validated for the determination of lumefantrine in human plasma. Lumefantrine and its internal standard halofantrine were extracted from plasma samples using protein precipitation with acetonitrile (0.2% perchloric acid) followed by solid-phase extraction with Hypersep C8 cartridges. Chromatographic separation was performed on a Zorbax SB-CN HPLC column (3.0 × 150 mm, 3.5 μm) with water/methanol (0.1% TFA) as the mobile phases in a gradient elution mode. Detection was performed using UV/vis detector at λ = 335 nm. The method showed to be linear over a range of 50-10,000 ng/mL with acceptable intra- and inter-day precision and accuracy. The mean recoveries were 88.2% for lumefatrine and 84.5% for the I.S. The internal standard halofantrine is readily available from commercial sources. This method was successfully applied to a pharmacokinetic interaction study between a first-line antimalarial combination (artemether—lumefantrine) and antiretroviral therapy.

scholarly journals Rapid and Sensitive Method for Quantitative Determination of Lopinavir and Ritonavir in Human Plasma by Liquid Chromatography- Tandem Mass Specrtometry

2009 ◽  
Vol 6 (1) ◽  
pp. 223-230 ◽  
Author(s):  
G. A. Temghare ◽  
S. S. Shetye ◽  
S. S. Joshi
Keyword(s):  
Liquid Chromatography ◽  
Human Plasma ◽  
Solid Phase ◽  
Internal Standard ◽  
Therapeutic Monitoring ◽  
Tandem Mass ◽  
Limit Of Quantification ◽  
Liquid Chromatography Tandem Mass ◽  
Sensitive Method

A rapid and sensitive liquid chromatography-mass spectrometric (LC-MS-MS) method for the simultaneous determination of lopinavir and ritonavir in human plasma using abacavir as internal standard has been developed and validated. Sample preparation of plasma involved solid phase extraction. Detection was performed using an Applied Biosystems Sciex API 2000 Mass spectrometer. The assay of lopinavir and ritonavir was linear over the range of 50 ng mL-1to 20000 ng mL-1and 20 ng mL-1to 3000 ng mL-1 respectively with a precision of <15% and accuracy in the range of 85-115%. The limit of quantification in plasma for lopinavir and ritonavir was 50 ng mL-1and 20 ng mL-1respectively. The described method has the advantage of being rapid and easy and it could be applied in therapeutic monitoring of these drugs in human plasma

scholarly journals Determination of Tianeptine in Tablets by High-Performance Liquid Chromatography with Fluorescence Detection

2007 ◽  
Vol 90 (3) ◽  
pp. 720-724
Author(s):  
Sevgi Tatar Ulu
Keyword(s):  
Lower Limit ◽  
High Performance ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Linear Calibration ◽  
Fluorescence Detector ◽  
Relative Standard ◽  
Validation Parameters ◽  
Limit Of Quantitation

Abstract A sensitive and selective high-performance liquid chromatographic method has been developed for the determination of tianeptine (Tia) in tablets. The method is based on derivatization of Tia with 4-chloro-7-nitrobenzofurazan (NBD-Cl). A mobile phase consisting of acetonitrile10 mM orthophosphoric acid (pH 2.5; 77 + 23) was used at a flow rate of 1 mL/min on a C18 column. The Tia-NBD derivative was monitored using a fluorescence detector, with emission set at 520 nm and excitation at 458 nm. Gabapentin was selected as an internal standard. Linear calibration graphs were obtained in the concentration range of 45300 ng/mL. The lower limit of detection (LOD) was 10 ng/mL at a signal-to-noise ratio of 4. The lower limit of quantitation (LOQ) was 45 ng/mL. The relative standard values for intra- and interday precision were &lt;0.46 and &lt;0.57%, respectively. The recovery of the drug samples ranged between 98.89 and 99.85%. No chromatographic interference from the tablet excipients was found. The proposed method was validated in terms of precision, robustness, recovery, LOD, and LOQ. All the validation parameters were within the acceptance range. The proposed method was applied for the determination of Tia in commercially available tablets. The results were compared with those obtained by an ultraviolet spectrophotometric method using t- and F-tests.

scholarly journals Determination of nimodipine in plasma by HPLC-MS/MS and pharmacokinetic application

2010 ◽  
Vol 46 (4) ◽  
pp. 665-677 ◽  
Author(s):  
Demétrius Fernandes do Nascimento ◽  
Manoel Odorico de Moraes ◽  
Fernando Antônio Frota Bezerra ◽  
Andréa Vieira Pontes ◽  
Célia Regina Amaral Uchoa ◽  
...  
Keyword(s):  
Human Plasma ◽  
Internal Standard ◽  
Pharmacokinetic Profile ◽  
Plasma Samples ◽  
Linear Calibration ◽  
Limit Of Quantification ◽  
Standard Curve ◽  
Liquid Liquid Extraction ◽  
Highly Sensitive

To develop and validate a rapid, specific and highly sensitive method to quantify nimodipine in human plasma using dibucaine as the internal standard (IS). The analyte and IS were extracted from plasma samples by liquid-liquid extraction using hexane-ethyl acetate (1:1 v/v). The chromatographic separation was performed on a Varian® Polaris C18 analytical column (3 μm, 50 x 2.0 mm) and pre-column SecurityguardTM C18 (4.0 x 3.0 mm) with a mobile phase of Acetonitrile-Ammonium acetate 0.02 ml/L (80:20v/v). The method had a chromatographic run time of 4.5 min and linear calibration curve over the range of 0.1- 40 ng/mL (r > 0.9938). The limit of quantification was 100 pg/mL. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. This validated method was successfully applied in determining the pharmacokinetic profile of nimodipine tablets of 30 mg administered to 24 healthy volunteers. The proposed method of analysis provided a sensitive and specific assay for nimodipine determination in human plasma. The time for the determination of one plasma sample was 4.5 min. This method is suitable for the analysis of nimodipine in human plasma samples collected for pharmacokinetic, bioavailability or bioequivalence studies in humans.

scholarly journals A Simple High-Performance Liquid Chromatography Method for Simultaneous Determination of Three Triazole Antifungals in Human Plasma

2012 ◽  
Vol 57 (1) ◽  
pp. 484-489 ◽  
Author(s):  
Mei Zhang ◽  
Grant A. Moore ◽  
Murray L. Barclay ◽  
Evan J. Begg
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Simultaneous Determination ◽  
Human Plasma ◽  
High Performance ◽  
Internal Standard ◽  
Gradient Elution ◽  
Limit Of Quantification ◽  
Chromatography Method

ABSTRACTA rapid and simple high-performance liquid chromatography (HPLC) assay was developed for the simultaneous determination of three triazole antifungals (voriconazole, posaconazole, and itraconazole and the metabolite of itraconazole, hydroxyitraconazole) in human plasma. Sample preparation involved a simple one-step protein precipitation with 1.0 M perchloric acid and methanol. After centrifugation, the supernatant was injected directly into the HPLC system. Voriconazole, posaconazole, itraconazole, its metabolite hydroxyitraconazole, and the internal standard naproxen were resolved on a C6-phenyl column using gradient elution of 0.01 M phosphate buffer, pH 3.5, and acetonitrile and detected with UV detection at 262 nm. Standard curves were linear over the concentration range of 0.05 to 10 mg/liter (r2> 0.99). Bias was <8.0% from 0.05 to 10 mg/liter, intra- and interday coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.05 mg/liter.

scholarly journals Determination of Dialkyl Phosphates as Breakdown Products of Organophosphorus Insecticides in Fruit Juices by HPTLC with Fluorescence Detection

2009 ◽  
Vol 92 (3) ◽  
pp. 691-698 ◽  
Author(s):  
Wolfgang Schwack ◽  
Tatjana Zeisler ◽  
Constanze Stiefel
Keyword(s):  
Apple Juice ◽  
Degradation Products ◽  
Organophosphorus Pesticides ◽  
Internal Standard ◽  
Barium Chloride ◽  
Fruit Juices ◽  
Limit Of Quantification ◽  
Dimethyl Phosphate ◽  
Relative Standard

Abstract Dialkyl phosphates (DAP) are common degradation products of organophosphorus pesticides that are used as urinary biomarkers for human exposure. An HPTLC method was developed for the quantitative determination of DAP in fruit juices, i.e., dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), diethyl phosphate (DEP), and diethyl thiophosphate (DETP). Dibutyl phosphate (DBP) was used as an internal standard. The method was based on precipitation of fruit acids in the presence of barium chloride and acetonitrile and liquidliquid extraction with acetonitrilediethyl ether. Extracted DAP were derivatized with 1-(bromoacetyl)pyrene (BAP), and the BAP derivatives separated on HPTLC amino plates with dichloromethane as the mobile phase. Densitometry was performed by measurement of fluorescence at 366/&gt;400 nm. The limit of quantification (LOQ) values were between 0.8 and 1.4 ng/zone. Fluorescence enhancement was achieved by dipping the plate into a paraffin oil solution, increasing the sensitivity and resulting in an LOQ of 0.50.6 ng/zone. Repeatabilities with relative standard deviations of 3.5 (n = 5, at 1520 ng/zone) and coefficients of correlation of 0.9999 were highly satisfactory for rapid trace analysis of DAP in the fruit juices by HPTLC. The mean recoveries from apple juice spiked at 0.5 mg/L were 74, 83, 70, and 57 for DMP, DEP, DMTP, and DETP, respectively. If an application volume of 5 L of apple juice extract was applied, the LOQ in apple juice was 300 g/L. However, this can be lowered by application of higher volumes (up to 50 L) or a more concentrated derivatization batch.

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