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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., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A simple, selective and stability-indicating high-pressure liquid chromatographic method was developed for the analysis of ribavirin. Chromatographic separation was achieved by using a CPS Hypersil cyano column (4.6 × 250 mm, 5 µm particle size) with isocratic elution of the mobile phase, which was composed of 50 mM phosphate buffer, adjusted at pH 4 with phosphoric acid. The mobile phase was pumped at a flow rate of 0.8 mL/min. The detector was set at 240 nm and quantification of the analyte was based on peak area measurement. The method was validated with respect to linearity, range, precision, accuracy, selectivity, robustness, limit of detection and limit of quantitation. The calibration curve was linear in the range of 5-200 µg/mL with correlation coefficient > 0.999. Ribavirin was subjected to forced degradation studies under two conditions: mild and extensive stress testing. These studies included the effects of hydrolysis (neutral, acidic and alkaline) and oxidation, photolysis and dry heat). The proposed method was proved to be stability-indicating by the resolution of the drug from its forced degradation products, making use of the diode array detector as a tool for confirmation of peak identity and purity. Moreover, the kinetics of alkaline degradation of ribavirin were investigated, an Arrhenius plot was constructed and the activation energy was calculated. The developed method was also extended to analyze ribavirin in capsules and in human plasma with good recovery values., (© The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

LC-MS/MS has been applied for the rapid determination of the nucleoside analogue ribavirin in human plasma and red blood cells. The incorporation of ribavirin to the erythrocytes has been assayed after in vitro incubation of the cells at different concentrations of the antiviral drug. After protein precipitation, samples were injected into a C8 column, achieving a complete separation of ribavirin from the endogenous isobaric compound uridine. Calibration ranges varied from 10 to 10,000 ng/mL in plasma and from 0.2 to 200 ng/cell pellet in red blood cells. Precision and accuracy values were always below 10 and 13%, respectively, in all assayed matrices. Ribavirin was demonstrated to remain unchanged after short and long time storage. No matrix effects could be assessed for the analyzed matrices. The developed method has been fully validated. Monitoring of ribavirin concentration in red blood cells in addition to the classic plasma monitoring of the drug could help to explain its efficacy and safety profiles in patients., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A rapid and sensitive quantitative assay method was developed for determining ribavirin pharmacokinetic in human plasma. The chromatographic separation was achieved within 4.5 min using a SinoChrom ODS-BP column (4.6 x 150 mm, 5 microm) with acetonitrile-water (1 mmol/L ammonium acetate buffer, 0.1% formic acid; 15:85, v/v) at a constant flow rate of 0.8 mL/min. The MRM pairs were m/z 245.2 --> m/z 113.1 for ribavirin and m/z 226.1 --> m/z 152.1 for acyclovir (internal standard), respectively, with dwell times of 200 ms for each transition. The results showed calibration curve for ribavirin was linear over a concentration range of 1-1000 ng/mL. The lower limit of quantification (LLOQ) was 1 ng/mL ribavirin. Twenty healthy volunteers received a 300 mg oral dose of ribavirin. Blood samples were then collected up to 120 h postdosing. All plasma data were comodeled for ribavirin by using noncompartmental modeling. The single dose of ribavirin was well tolerated and no serious adverse effects occurred. The mean time to maximum concentration was about 1.25 h. The mean maximum concentration of drug in plasma for oral ribavirin was 250 ng/mL. The mean elimination half-life was 43.6 h. The present study describes a simple, specific, sensitive HPLC-MS/MS method for measuring plasma drug concentration and analyzing human pharmacokinetics of ribavirin.

Ribavirin (RBV) in combination with pegylated interferon is the current standard treatment for chronic hepatitis C. Exposure to RBV seems crucial for achieving the best virologic response. However, RBV may cause anemia in a dose-dependent manner. Therefore, monitoring RBV plasma levels could be useful for individual tailoring of RBV dosing. A rapid assay was developed to quantify RBV using high-performance liquid chromatography and ultraviolet detection. Extraction of RBV from plasma was performed using a novel method based on ultrafiltration in one step that allows direct injection into the high-performance liquid chromatography without any prior steps of dryness or reconstitution. The method was validated over the range of 0.05 to 5.0 microg/mL following the EMEA Validation of Analytical Procedures (CPMP/ICH/281/95) recommendations. The clinical interest of this assay was evaluated in a subset of patients under RBV therapy. Mean RBV plasma concentrations at steady state were higher in responders compared with nonresponders (3-3.2 mug/mL versus 2.2-2.3 microg/mL).

A simple high-performance liquid chromatography method for the determination of the antiviral agent ribavirin in human plasma was developed and validated. The method involved solid-phase extraction on phenyl boronic acid cartridges, a reversed-phase liquid chromatography with a Waters Atlantis dC18 (150 mm x 3.9 mm, 5 microm) column and a mobile phase consisting of 10 mM potassium phosphate buffer (pH 4.0), and ultraviolet detection at 207 nm. This assay proved to be sensitive (lower limit of quantification of 0.05 microg/ml), linear (correlation coefficients >or=0.997), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation

For the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous determination of ribavirin and rabavirin base was developed and validated over the concentration range of 10-5,000 ng/ml, respectively, using a 0.025 ml monkey plasma sample. Ribavirin, ribavirin base, and the internal standards were extracted from monkey plasma via protein precipitation. After evaporation of the supernatant, the extract was reconstituted with 5% methanol (containing 0.1% formic acid) and injected onto the LC-MS/MS system. Optimum chromatographic separation was achieved on a Waters Atlantis dc18 (150 mm x 2.1mm, 5 microm) column with mobile phase run in gradient with 100% water containing 0.5% formic acid (A) and 90% acetonitrile (containing 0.5% formic acid (B). The flow rate was 0.4-0.6 ml/min with total cycle time of approximately 7.0 min. Post-column addition of acetonitrile (containing 0.1% formic acid) at 0.3 ml/min was used to increase the ionization efficiency in the MS source. The method was validated for sensitivity, linearity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The intra-day and inter-day precision and accuracy of the quality control (QC) samples were <9.0% relative standard deviation (R.S.D.) and 10.8% bias for ribavirin, and 10.3% R.S.D. and 11.3% bias for ribavirin base. The current specific, accurate and precise assay is useful in support of the toxicokinetic and pharmacokinetic studies of these compounds.

Measurement of ribavirin plasma levels in HCV-positive patients have been shown to be useful in order to optimise individual ribavirin exposure. Efficacy and toxicity of this drug are shown to be concentration-dependant. A simple HPLC-UV method was developed and validated, which has an easy liquid/liquid extraction, sensitive limit of detection, without any interference peaks, reproducible and linear over the range of clinical relevant concentrations. The assay warrants further evaluation as a tool for ribavirin therapeutic drug monitoring in HCV-positive patients.

Viramidine is a prodrug of ribavirin. To facilitate pharmacokinetics studies of viramidine in man, a sensitive and specific LC-MS/MS method for the simultaneous analyses of viramidine and ribavirin in human plasma was developed and validated. The method involved the addition of [13C]viramidine and [13C]ribavirin as internal standards, protein precipitation with acetonitrile, HPLC separation, and quantification by MS/MS system using positive electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor-->product ion transitions were monitored at 245-->113, 250-->113, 244-->112, and 249-->112 for ribavirin, [13C]ribavirin, viramidine, and [13C]viramidine, respectively. The calibration curves for viramidine and ribavirin were linear over a concentration range of 1-1000 ng/mL. For both viramidine and ribavirin, the lower limit of quantification (LLOQ) was 1 ng/mL. For viramidine, intra- and inter-day analyses of QC samples at 1, 5, 250, and 1000 ng/mL indicated good precision (%CV between 1.0 and 7.0%) and accuracy (%bias between -4.3 and 5.2%). For ribavirin, intra- and inter-day analyses of QC samples at 1, 5, 250, and 1000 ng/mL indicated similar precision (%CV between 0.8 and 8.3%) and accuracy (%bias between -5.8 and 9.4%). Both viramidine and ribavirin were stable in human plasma stored at room temperature for at least 3 h, 4 degrees C for at least 6 h, and for at least three freeze-thaw cycles. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of viramidine and ribavirin in man following administration of viramidine.

A sensitive and specific method using high-performance liquid chromatography (LC)-tandem mass spectrometry (MS) for the analysis of total ribavirin in human red blood cells (RBC) is developed and validated. The method involves the addition of an internal standard and perchloric acid, the conversion of ribavirin phosphorylated metabolites to ribavirin, purification with a solid-phase exchange cartridge, and LC-MS-MS analysis. The MS-MS is selected to monitor m/z 245-113 for ribavirin and m/z 250-113 for [13C]ribavirin using positive electrospray ionization. The calibration curve is linear over a concentration of 100-10,000 ng/mL with a limit of quantitation of 100 ng/mL. Mean interassay accuracy for quality control (QC) at 100, 1000, and 10,000 ng/mL are 101.8%, 99.4%, and 98.8%, respectively. Mean interassay precision (%CV) for QC at 100, 1000, and 10,000 ng/mL are 5.0%, 5.0%, and 2.5%, respectively. Extractibility of total ribavirin from RBC is confirmed with RBC obtained from a [(14)C]ribavirin-dosed monkey. The method is used to determine the free and total ribavirin concentration in human RBC obtained from hepatitis C patients treated with ribavirin.

Ribavirin is a purine nucleoside analog with broad spectrum activity against a spectrum of DNA and RNA viruses. To facilitate pharmacokinetics studies, a LC-MS-MS method for the analysis of ribavirin in rat and monkey plasma was developed and validated. The method involved the addition of acyclovir as an internal standard and protein precipitation with acetonitrile followed by separation by an Intertsil Silica column and quantification by a MS-MS equipped with a positive electrospray ionization in the multiple reaction monitoring mode. The MS-MS reaction was selected to monitor the 245-->113 and 226-->152 transitions for ribavirin and internal standard, respectively. The calibration curve was linear over a concentration range of 10-5000 ng/ml. The lower limit of quantitation was 10 ng/ml, the coefficient of variation (CV) was 8-11%, and the bias was 1-3%. Intra-day and inter-day analysis of QC samples at 30, 1500 and 3500 ng/ml indicate that the method was precise (CV<18%) and accurate (bias<13%). Ribavirin in rat and monkey plasma was stable at 5 degrees C for at least 24 h, 0 degrees C for at least 4 h, and after three freeze-thaw cycles. This specific, accurate and precise assay is useful in the study of the pharmacokinetics of this compound.

A rapid assay for determination of ribavirin in serum using solid-phase extraction (SPE), high-performance liquid chromatography (HPLC), and UV-detection was developed. The SPE uses phenylboronic acid columns with an approximately 100% recovery for ribavirin. The concentration-peak area relation was linear (r > 0.995), from 1 to 64 microM in 100 microL serum. The limit of detection was 0.1 microM. The intraassay CV was 3.2% at treatment levels (9.7 microM) and 11.5% at 0.4 microM. The method is used to monitor patients undergoing ribavirin treatment for hepatitis C (HCV). Samples from HCV-infected patients with and without renal dysfunction have been analyzed without interference of endogenous compounds. It is concluded that the method is useful for routine therapeutic drug monitoring.