Validated RP-HPLC Method for Simultaneous Determination of Ribavirin, Sofosbuvir and Daclatasvir in Human Plasma: A Treatment Protocol Administered to HCV Patients in Egypt

2019 ◽  
Vol 57 (7) ◽  
pp. 636-643 ◽  
Author(s):  
Aya A Youssef ◽  
N Magdy ◽  
Lobna A Hussein ◽  
A M El-Kosasy
Keyword(s):  
Human Plasma ◽  
Method Validation ◽  
Treatment Protocol ◽  
Internal Standard ◽  
Gradient Elution ◽  
Hplc Method ◽  
Array Detector ◽  
Therapeutic Drug ◽  
Bioanalytical Method Validation ◽  
Rp Hplc

Abstract Egypt has the highest prevalence of hepatitis C virus (HCV) in the world thus it launched a national program for eliminating HCV aiming to treat 300,000 HCV patients per year. Three anti-HCV co-administered drugs; ribavirin (RBV), sofosbuvir (SF) daclatasvir (DAC) were simultaneously determined in human plasma by a validated, simple and sensitive RP-HPLC method using propyl paraben as an internal standard. Liquid–liquid extraction using ethyl acetate was used for samples extraction. Chromatographic separation was achieved on Scharlau® C18 column (250 × 4.6 mm2, 5 μm). Gradient elution was employed with a mobile phase mixture of water and acetonitrile at a flow rate 1 mL/min. UV detection using photodiode array detector was carried out at 207, 260 and 312 nm for RBV, SF and DAC, respectively. Method validation was performed according to the FDA guidelines for bioanalytical method validation. The calibration curves were linear over the ranges (0.5–80, 0.1–40 and 0.5–80 μg/mL) with average recoveries (100.64–108.28%, 98.48–105.91% and 97.68–101.38%) for RBV, SF and DAC, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limits. Stability assays revealed that the three studied analytes were stable during sample storage, preparation and injection. The method can be successfully applied in routine analysis of plasma of HCV patients treated with this combination therapy which aids in therapeutic drug monitoring and patients’ follow-up especially in Egypt and other developing countries fighting HCV.

scholarly journals BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF SOFOSBUVIR FROM HUMAN PLASMA

2017 ◽  
Vol 9 (3) ◽  
pp. 35 ◽  
Author(s):  
S. Madhavi ◽  
A. Prameela Rani
Keyword(s):  
Human Plasma ◽  
Method Development ◽  
Quantitative Estimation ◽  
Internal Standard ◽  
Hplc Method ◽  
Array Detector ◽  
Therapeutic Drug ◽  
Specificity And Sensitivity ◽  
Photodiode Array Detector ◽  
Development And Validation

Objective: This study points to build up and validate a simple methodology to quantify the most used drug sofosbuvir for the treatment of hepatitis C virus (HCV) infection, in human plasma by using atazanavir as an Internal Standard (IS) for preclinical studies and validate as per USFDA guidelines.Methods: Sofosbuvir was isolated from plasma samples by liquid-liquid extraction method using acetonitrile; good chromatographic separation was achieved on Kromasil Column (250 mm ×4.6 mm, 5 µm). The mobile phase consisted of 0.1 % orthophosphoric acid (OPA) buffer pH 2 and acetonitrile in the ratio of (68:32, v/v), respectively. The analysis time was 7 min at a flow rate 1 ml/min. The photodiode array detector (PDA) detection was carried out at 228 nm. The suggested method was validated by performing linearity, system suitability, specificity and sensitivity, accuracy and precision, recovery, ruggedness, stability studies. The method was validated as per USFDA guidelines.Results: The developed method resulted in retention times of sofosbuvir and IS were found out to be 4.7 and 4.2 min respectively. The calibration curves are linear (r2 = 0.999) over the concentration range of 0.050-2.0 µg/ml of plasma analytes concentration. LOQ value was found to be 0.050 µg/ml with precision and accuracy. Within-batch % mean accuracy of the method ranged between 96.00% and 109.09%, and within-batch and total precision, expressed as the coefficient of variation, was 1.40–10.33%. Overall percentage mean recovery of sofosbuvir from spiked plasma was 84.14%. All the validated parameters were found to be within the limit.Conclusion: A simple, accurate, precise, linear, rugged and rapid RP-HPLC method was developed for quantitative estimation of sofosbuvir in human plasma and should be suitable for conducting pharmacokinetics studies and therapeutic drug monitoring.

scholarly journals Development and Validation of a Method for Quantification of Favipiravir as COVID-19 Management in Spiked Human Plasma

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3789
Author(s):  
Mohammad Hailat ◽  
Israa Al-Ani ◽  
Mohammed Hamad ◽  
Zainab Zakareia ◽  
Wael Abu Dayyih
Keyword(s):  
Human Plasma ◽  
Internal Standard ◽  
Weighting Factor ◽  
Hplc Method ◽  
Bioanalytical Method Validation ◽  
Spiked Human Plasma ◽  
Fda Guidance ◽  
Significant Difference ◽  
Us Fda ◽  
Carry Over

In the current work, a simple, economical, accurate, and precise HPLC method with UV detection was developed to quantify Favipiravir (FVIR) in spiked human plasma using acyclovir (ACVR) as an internal standard in the COVID-19 pandemic time. Both FVIR and ACVR were well separated and resolved on the C18 column using the mobile phase blend of methanol:acetonitrile:20 mM phosphate buffer (pH 3.1) in an isocratic mode flow rate of 1 mL/min with a proportion of 30:10:60 %, v/v/v. The detector wavelength was set at 242 nm. Maximum recovery of FVIR and ACVR from plasma was obtained with dichloromethane (DCM) as extracting solvent. The calibration curve was found to be linear in the range of 3.1–60.0 µg/mL with regression coefficient (r2) = 0.9976. However, with acceptable r2, the calibration data’s heteroscedasticity was observed, which was further reduced using weighted linear regression with weighting factor 1/x. Finally, the method was validated concerning sensitivity, accuracy (Inter and Intraday’s % RE and RSD were 0.28, 0.65 and 1.00, 0.12 respectively), precision, recovery (89.99%, 89.09%, and 90.81% for LQC, MQC, and HQC, respectively), stability (% RSD for 30-day were 3.04 and 1.71 for LQC and HQC, respectively at −20 °C), and carry-over US-FDA guidance for Bioanalytical Method Validation for researchers in the COVID-19 pandemic crisis. Furthermore, there was no significant difference for selectivity when evaluated at LLOQ concentration of 3 µg/mL of FVIR and relative to the blank.

scholarly journals DEVELOPMENT AND VALIDATION OF BIOANALYTICAL HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF CILNIDIPINE AND NEBIVOLOL IN HUMAN PLASMA

2017 ◽  
Vol 9 (10) ◽  
pp. 253
Author(s):  
Aruna G. ◽  
Bharathi K ◽  
Kvsrg Prasad
Keyword(s):  
Human Plasma ◽  
Analytical Method ◽  
Internal Standard ◽  
Hplc Method ◽  
Pharmacokinetic Parameters ◽  
Simultaneous Estimation ◽  
Time Data ◽  
Bioanalytical Method Validation ◽  
Limit Of Quantitation ◽  
The Mean

Objective: To develop and validate a modified isocratic reversed-phase high performance liquid chromatographic (RP-HPLC) method for determination of cilnidipine and nebivolol in human plasma to be used for pharmacokinetic studies.Methods: The drug was extracted from plasma samples by direct protein precipitation technique using acetonitrile. Amlodipine was used as internal standard (IS). Samples were analyzed on BDS C18 column (250 x 4.6 mm, 5 µm), applying ortho phosphoric acid (0.1%): Acetonitrile, at a ratio of 45:55 v/v in isocratic mode as a mobile phase at a flow rate of 1 ml/min to attain adequate resolution. Separations were performed at room temperature and monitored at a wavelength of 260 nm after injection of 50μl samples into the HPLC system. The analytical method was validated according to FDA bioanalytical method validation guidance. The method was applied for pharmacokinetic study of cilnidipine and nebivolol tablets-10 mg and 5 mg were administered as a single dose to 6 healthy male rabbits under fasting condition. Twelve blood samples were withdrawn from each rabbit over 24 h periods. From the plasma concentration-time data of each individual, the pharmacokinetic parameters; Cmax, Tmax, AUC0-t and AUC0-∞ were calculated.Results: A peak area was obtained for cilnidipine and nebivolol at 3.943 and 4.719 min retention time respectively. Linearity was established at a concentration range of 0.20-20 μg/ml (r2=0.999, n=8) for cilnidipine and 0.02-2 μg/ml (r2=0.999, n=8) for nebivolol. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 0.2μg/ml for cilnidipine and 0.02 μg/ml for nebivolol. The coefficients of variation (%cv) of the intra-day and inter-day precision of cilnidipine at 600, 1000 and 1600ng/ml levels were found to be 6.90%, 6.19%, 5.22%; and 7.74%, 6.54%, 5.77%, respectively, which are lower than the accepted criteria limits (15-20 %). The mean recovery (%) cilnidipine at 600, 1000, and 1600ng/ml was found to be 101.03%, 99.27% and 104.87%, and for nebivolol 60, 100, and 160 ng/ml was found to be 106.13%, 107.03% and 98.06% respectively. Stability at different conditions and in autosampler was also established. The mean pharmacokinetic parameters; Cmax, Tmax, AUC0-t and AUC0-∞ were 6 ng/ml, 2 hr, 96.76 mg. hr/ml, 63.45 mg. hr/ml for cilnidipine and 5.8ng/ml, 2hr, 74.78 mg. hr/ml, 100.25 mg. hr/ml for nebivolol respectively.Conclusion: The present analytical method was found to be specific, sensitive, accurate and precise for quantification of cilnidipine and nebivolol in human plasma. It can be successively applied for pharmacokinetics, bioavailability and bioequivalence studies.

scholarly journals Development and Validation of RP-HPLC Method for Azilsartan Medoxomil Potassium Quantitation in Human Plasma by Solid Phase Extraction Procedure

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Paras P. Vekariya ◽  
Hitendra S. Joshi
Keyword(s):  
Solid Phase Extraction ◽  
Human Plasma ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Hplc Method ◽  
Array Detector ◽  
Phase Extraction ◽  
Rp Hplc ◽  
Limit Of Quantitation ◽  
Azilsartan Medoxomil

Simple and rapid reverse phase high-performance liquid chromatography (RP-HPLC) method was developed and validated using solid phase extraction (SPE) technique for the determination of Azilsartan Medoxomil Potassium (AMP) in human plasma; detection was carried out by photo diode array detector. Chromatographic separation of the analyte AMP was achieved within 7.5 min by Waters symmetry C18 (4.6 × 250 mm, 5 µm) column, mobile phase was 25 mM ammonium acetate buffer (pH 5.5): acetonitrile 55 : 45 v/v, flow rate was 1.0 mL/min, and the detection was carried out at 254 nm. Calibration curve was linear (r2 > 0.9985) in the range of 1.0–9.0 µg/mL, limit of detection (LOD) and limit of quantitation (LOQ) were 0.150 µg/mL and 0.400 µg/mL, respectively, and intra- and interday deviations were between 1.53–8.41% and 1.78–4.59%, respectively. The overall mean recovery of AMP was 92.35%. No any endogenous constituents were found to interfere at retention time of the analyte. This new RP-HPLC method was successfully validated and may be applied to conduct bioavailability and bioequivalence studies of AMP.

scholarly journals Simultaneous Quantification of Methotrexate and Its Metabolite 7-Hydroxy-Methotrexate in Human Plasma for Therapeutic Drug Monitoring

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Xinxin Ren ◽  
Zhipeng Wang ◽  
Yunlei Yun ◽  
Guangyi Meng ◽  
Xialan Zhang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Therapeutic Drug Monitoring ◽  
Human Plasma ◽  
Drug Monitoring ◽  
Internal Standard ◽  
Gradient Elution ◽  
Therapeutic Drug ◽  
Tandem Mass ◽  
The Matrix ◽  
Analytical Time

Objective. To establish and validate a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of methotrexate (MTX) and its major metabolite 7-hydroxy-methotrexate (7-OH-MTX) in human plasma. Method. The chromatographic separation was achieved on a Zorbax C18 column (3.5 μm, 2.1 × 100 mm) using a gradient elution with methanol (phase B) and 0.2% formic acid aqueous solution (phase A). The flow rate was 0.3 mL/min with analytical time of 3.5 min. Mass spectrometry detection was performed in a triple-quadruple tandem mass spectrometer under positive ion mode with the following mass transitions: m/z 455.1/308.1 for MTX, 471.0/324.1 for 7-OH-MTX, and 458.2/311.1 for internal standard. The pretreatment procedure was optimized with dilution after one-step protein precipitation. Results. The calibration range of methotrexate and 7-OH-MTX was 5.0-10000.0 ng/mL. The intraday and interday precision and accuracy were less than 15% and within ±15% for both analytes. The recovery for MTX and 7-OH-MTX was more than 90% and the matrix effect ranged from 97.90% to 117.60%. Conclusion. The method was successfully developed and applied to the routine therapeutic drug monitoring of MTX and 7-OH-MTX in human plasma.

Development and Validation of an RP-HPLC Method for Determination of Atorvastatin and its Hydroxyl Metabolites in Human Plasma

2020 ◽  
Vol 16 (3) ◽  
pp. 238-245
Author(s):  
Dagmara Sowińska ◽  
Alicja Pogorzelska ◽  
Marlena Rakicka ◽  
Justyna Sznura ◽  
Justyna Janowska ◽  
...  
Keyword(s):  
Human Plasma ◽  
Internal Standard ◽  
Hplc Method ◽  
Active Metabolites ◽  
Peak Separation ◽  
Rp Hplc ◽  
Increased Risk ◽  
Relative Standard ◽  
Development And Validation

Background: Atorvastatin (AT) belongs to cholesterol-lowering agents, commonly used in patients with an increased risk of cardiovascular disease. The drug, as well as its hydroxyl metabolites, exhibit pharmacological activity, and their plasma levels may be helpful in the assessment of the therapeutic effectiveness. Objective: Development and validation of a fast and reproducible RP-HPLC method with UV detection for the simultaneous determination of atorvastatin and its active metabolites, para-hydroxy-atorvastatin (p-OH-AT) and ortho-hydroxy-atorvastatin (o-OH-AT) in human plasma. Methods: Optimal conditions of chromatographic separation of the analytes, as well as rosuvastatin, chosen as an internal standard, were studied. The absorbance of the compounds was measured at λ=248 nm. Validation of the method was performed. The usefulness of the method was confirmed for determination of the analytes in plasma of patients treated with the drug. Results: Total peak separation was achieved at LiChrospher 100 RP-18 column with a mobile phase composed of methanol and water (1:1,v:v) and a flow rate of 1.2 ml/min. The method was linear in the ranges of 0.025 - 1.0 μg/ml for AT, o-OH-AT and p-OH-AT. Intra- and inter-assay precision expressed as relative standard deviation was ≤13% for AT, ≤12% for p-OH-AT and ≤11% for o-OH-AT. Intraand inter-day accuracy of the method, expressed as a relative error, was ≤15%. Conclusion: The elaborated HPLC method is specific, repeatable, reproducible, adequately accurate and precise and fulfills the validation requirements for the bioanalytical method. The method was successfully applied for analysis of atorvastatin and its o-hydroxy metabolite in plasma of patients treated with the drug.

scholarly journals DEVELOPMENT AND VALIDATION OF A SIMPLE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY–UV METHOD FOR THE DETERMINATION OF VINORELBINE - A CHEMOTHERAPEUTIC DRUG IN SPIKED HUMAN PLASMA

2019 ◽  
Vol 12 (1) ◽  
pp. 369
Author(s):  
Useni Reddy Mallu ◽  
Venkateswara Rao Anna ◽  
Bikshal Babu Kasimala
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Internal Standard ◽  
Hplc Method ◽  
Pharmacokinetic Studies ◽  
Chemotherapeutic Drug ◽  
Spiked Human Plasma ◽  
Rp Hplc

Objective: Vinorelbine (VNRB) is a chemotherapeutic drug used to treat non-small cell lung cancer and breast cancer. Literature survey reveals that there are no reverse-phase high-performance liquid chromatography (RP-HPLC) methods reported for the estimation of VNRB in spiked human plasma. Hence, the present work aimed to develop a simple and efficient RP-HPLC method for the estimation of VNRB in human plasma.Methods: Specimen preparation for the measurement of VNRB was performed through liquid-liquid extraction using methanol as extracting solvent and reconstructed with mobile phase. Paclitaxel (PCTX) was used as internal standard. HPLC method was optimized and validated as per the US FDA bioanalytical guidelines. VNRB and internal standard were separated on Kromasil® C18 (250×4.6 mm; id 5 μ) using acetate buffer (pH=5.9) and methanol in the ratio of 85:15 (v/v) at 1 ml/min flow rate. Eluted compounds were recorded using UV detector at 235 nm.Results: The retention time of PCTX and internal standard was found to be 4.3 and 9.0 min, respectively. The analytical measuring ranges were found to be 5–750 ng/ml (r2>0.9998). The method was found to be simple, accurate, precise, and stable and there is no interference of plasma matric components.Conclusion: The described HPLC method allows for the measurement of total and free PCTX in both plasma and cord blood and can utilize for the estimation of drug in pharmacokinetic studies.

scholarly journals Development and Validation of RP-HPLC Method for Desoximetasone in Bulk and Cream Formulation

2019 ◽  
Vol 9 (3) ◽  
pp. 154-159
Author(s):  
Ashish Gorle ◽  
Jayashri Mahajan ◽  
Prathamesh Bhave
Keyword(s):  
Method Validation ◽  
Mobile Phase ◽  
High Performance ◽  
Gradient Elution ◽  
Hplc Method ◽  
Topical Steroids ◽  
Cream Formulation ◽  
Rp Hplc ◽  
Validation Parameters ◽  
Development And Validation

Desoximetasone chemically is 9-fluoro-IIβ21-dihydroxy-I6a-methylpregna-I.4-diene-3.The precise mechanism of the anti-inflammatory activity of topical steroids in the treatment of steroid-responsive dermatoses, in general, is uncertain. So, in present investigation chromatographic methods were developing use RP-HPLC for estimation of Desoximetasone in bulk and in cream formulation and method validation according to ICH guidelines. The main objective of this study was to develop a simple and reproducible method for desoximetasone by Reverse Phase High Performance Liquid Chromatography (RP-HPLC). In this work the desoximetasone separation was carried out by using C18 cosmosile column (250mmx4.6mm particle size 5µm). By using 0.1% orthrophosphoric acid pH adjusted up to 3 at uv detection of 240nm.The mobile phase was used at various ratio for gradient elution the ratio of mobile phase was 20:80 v/v. Methanol and water used for mobile phase and flow rate was being set at 1mL/min. The linearity of proposed method was found in range of r =0.9989. Statistically validation parameters such as linearity, accuracy, precision, LOD and LOQ were checked. Keywords: Desoximetasone, RP-HPLC, Method validation.

scholarly journals Bioanalytical Method Development and Validation for the Determination of Favipiravir in Spiked Human Plasma by using RP-HPLC

2021 ◽  
pp. 275-281
Author(s):  
Pallavi V. Duse ◽  
Kamalkishor G. Baheti
Keyword(s):  
Human Plasma ◽  
Hplc Method ◽  
Therapeutic Drug ◽  
Pharmacokinetic Studies ◽  
Gradient System ◽  
Spiked Human Plasma ◽  
Rp Hplc ◽  
Relative Standard ◽  
Limit Of Quantitation

A precise, simple and reproducible reverse phase liquid chromatography (RP-HPLC) method was developed and validated for determination of Favipiravir by using Carbamazepine as internal standard in spiked human plasma. A chromatographic separation was accomplished with Cromasil C18 (250mm x 4.6ID, Particle size: 5 micron) column using mobile phase consists of methanol: water in the ratio (35:65, %v/v), at pH 3.0 with binary gradient system-maintained flow rate at 0.8ml/min. The detection wavelength of drug sample was at 225 nm. Extraction was done by using ethyl acetate as extracting solvent. The retention time of Favipiravir was found to be 6.62 min.  The method was found to be linear in the concentration range of 0.2-3.2 µg/ml. Limit of quantitation (LOQ) value was found to be 0.72. The intra- and inter day precision and accuracy lies within the specified range. The recovery studies were found to be in the range of 97.6 to 100.2%. %Relative standard deviation (RSD) was found to be in the range of 0.07-2.80%. All parameters were found to be validated from spiked human plasma. The proposed RP-HPLC method is highly accurate and rapid for the determination of favipiravir in human plasma and can be applied for pharmacokinetic studies and Therapeutic drug monitoring.

DEVELOPMENT OF LIQUID-LIQUID EXTRACTION PROCEDURE AND ITS APPLICATION FOR QUANTIFICATION OF ERLOTINIB IN SPIKED HUMAN PLASMA USING RP-HPLC WITH UV DETECTION

INDIAN DRUGS ◽  
2016 ◽  
Vol 53 (06) ◽  
pp. 46-50
Author(s):  
A Faizee ◽  
◽  
S. S Sonawane ◽  
A. S. Patil ◽  
S. J Kshirsagar ◽  
...  
Keyword(s):  
Human Plasma ◽  
Ammonium Acetate ◽  
Extraction Procedure ◽  
Internal Standard ◽  
Hplc Method ◽  
Liquid Extraction ◽  
Calibration Model ◽  
Spiked Human Plasma ◽  
Liquid Liquid Extraction ◽  
Rp Hplc

A simple, rapid and accurate RP-HPLC method was developed and validated for the quantification of Erlotinib in spiked human plasma using liquid-liquid extraction. Sufficient recovery was obtained when drug and internal standard (Nabumetone) were extracted using ethyl acetate and 1N NaOH. Chromatographic separation was performed on C18 Phenomenex Hyperclone column (250 × 4.6 mm, 5 μm) using mobile phase acetonitrile: 20 mM ammonium acetate buffer pH 4.6 (60:40%,V/V). Flow rate was kept constant at 1 mL/min and detection was carried out at 331 nm. Calibration curve was found to be linear in the range of 100-3200 ng/mL. During the calibration experiments, it was found that heteroscedasticity can be minimized using weighted regression calibration model with weighing factor of 1/x2.

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