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Ribavirin is a synthetic, broad-spectrum antiviral drug. Ribavirin is recommended as an antiviral drug in the Interim Guidance for Diagnosis and Treatment (the seventh edition) of COVID-19. The ribavirin levels in red blood cells may be closely related to both its efficacy and adverse drug reactions. In this study, a simple and fast HPLC-UV method was established to determine the concentrations of total ribavirin in the red blood cells of 13 patients with COVID-19. Phosphorylated ribavirin was dephosphorylated by phosphatase incubation to obtain the total amount of ribavirin in red blood cells. The chromatographic column was an Atlantis C 18 . The recoveries were 85.45-89.05% at three levels. A good linear response was from 1 to 200 μg/ml, with a correlation coefficient of r 2  = 0.9991. The concentration of total ribavirin in the red blood cells of the patients ranged from 30.83 to 133.34 μg/ml. The same samples without phosphatase incubation ranged from 4.07 to 20.84 μg/ml. About 85% of ribavirin was phosphorylated in red blood cells. In addition, we observed changes in these patients' hematological parameters and found that the erythrocyte, hemoglobin and hematocrit declined to the lowest levels on the fifth day after discontinuation of ribavirin (p < 0.05)., (© 2022 John Wiley & Sons, Ltd.)

Ribavirin (RBV) is an effective antiviral drug, whose use is prohibited in animal husbandry worldwide. In this work, a novel immunizing hapten of RBV, named Hapten 4, was designed by comparing the conformational and electronic properties of RBV and haptens based on computational chemistry. Hapten 4 was synthesized and conjugated with carrier proteins to produce monoclonal antibody (mAb). The obtained mAb 4C3 for RBV exhibited an IC 50 value of 6.24 ng/ml in an indirect competitive enzyme-linked immunosorbent assay (icELISA) and displayed no cross-reaction with five other antiviral drugs, including amantadine. The applicability of the developed icELISA was verified in chicken, with a calculated limit of detection of 4.23 µg/kg. The recoveries in spiked chicken were 79.2%-107.3% with a coefficient of variation less than 15.9%. The results indicated that the produced antibody from the new hapten was reliable and would be useful for RBV screening in chicken. PRACTICAL APPLICATION: RBV is a broad-spectrum antiviral drug, which is commonly used illegally in poultry farms. A high-affinity mAb 4C3 against RBV was produced and used to develop icELISA with acceptable sensitivity and accuracy. The constructed icELISA has excellent performance for detecting RBV residues in chicken., (© 2021 Institute of Food Technologists®.)

Objectives: In the present study, an eco- friendly micellar liquid chromatographic technique was validated for separation and quantification of two drugs; namely ribavirin (RIV), and sofosbuvir (SBV) in pure form, pharmaceuticals containing them, human plasma and human urine. These drugs are administered co-administered for treatment of Hepatitis C virus (HCV) that causes hepatitis C in humans., Material and Methods: These drugs were separated using Nucleosil 100-5 phenyl column. Sodium dodecyl sulphate (SDS) solution (0.05M, pH 7.0) containing triethylamine (0.3%) and n-butanol (10%) was used as a mobile phase with 1.2 mLmin -1 flow rate and 215nm detection wavelength. Nine minutes were required for resolving the two drugs from the matrix., Results: The method showed good linearity for RIV and SBV with correlation coefficients (r 2 ) more than 0.9996 within the concentration ranges of (20-400) and (40-400) ngmL -1 in pure form, (30-300) and (50-300) ngmL -1 in human plasma and (20-400) and (40-400) ngmL -1 in human urine, respectively., Conclusion: The recommended method was applied for examination of RIV and SBV in pure and pharmaceuticals. The obtained results were statistically matched with reported methods with no significant differences. Also, the recommended method was effectively applied for estimation of both drugs in spiked human urine and plasma without purification or extraction steps and real samples of plasma and urine of humans having therapy of RIV and SBV, as well as, performing tablets dissolution-rate tests with satisfactory results., (Copyright © 2020 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.)

A novel magnetic molecularly imprinted polymer adsorbing material was successfully synthesized to detect ribavirin in animal feedstuff. Molecularly imprinted polymer was prepared through surface polymerization by using ribavirin as template molecule, methyl methacrylate, and γ-methacryloxypropyl trimethoxy silane functionalized magnetic mesoporous silica as bifunctional monomers, and ethylene diglycidyl ether as crosslinking agent. The prepared magnetic molecularly imprinted polymer was characterized by scanning electron microscopy and infrared spectroscopy. Static and dynamic adsorption experiments and selective adsorption analysis were performed to evaluate the adsorption and selectivity of magnetic molecularly imprinted polymer. Different experiments were conducted to optimize the magnetic solid-phase extraction conditions. Under optimal experimental conditions, a magnetic molecularly imprinted solid-phase extraction coupled with high-performance liquid chromatography method was successfully developed for ribavirin detection. The established method achieved a satisfactory linear range of 0.20-50 mg/L (R 2  > 0.99) and a low detection limit (0.081 mg/kg). An average recovery of 92-105% with relative standard deviation of <6.5% was obtained upon the application of the developed method to detect ribavirin in real feedstuff samples. Thus, established method can be used for the rapid and simple separation and detection of added ribavirin in feedstuff., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

For the first time, a magnetic solid-phase extraction with high-performance liquid chromatography detection method using Zr functionalized Fe 3 O 4 magnetic material to enrich ribavirin was successfully established. Zr components that modified in Fe 3 O 4 nanoparticles via a simple one-step hydrothermal method was selected in this work to specifically capture ribavirin by the strong chemical bonding between Zr components of Zr functionalized Fe 3 O 4 magnetic material and cis-hydroxyl of ribavirin, which was confirmed by pseudo-second-order kinetic model. And Fe 3 O 4 components were selected in this work to achieve simple operation. Under the optimal experimental conditions, proposed magnetic solid-phase extraction with high-performance liquid chromatography detection method along with Zr functionalized Fe 3 O 4 magnetic material offered a wide range linearity at 10-200 µg/L with correlation coefficient of 0.9978 with low detection limit of 2.68 µg/L for ribavirin. The relative standard deviations obtained from nine parallel extractions of 100 µg/L ribavirin were 4.41% and revealed good repeatability. This established method was successfully applied to detect real samples including chicken liver, egg, and shrimp with satisfactory recoveries of 74.13-92.9%., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this work, high affinity polyclonal antibodies for ribavirin (RBV) from new haptens were prepared and were used to analyse RBV residues in chicken muscle, eggs and duck muscle. The new haptens were synthesised with different spacers, and the best antibody was obtained with an IC 50 value as low as 0.61 ng/mL in indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivities with another five antiviral drugs including amantadine, rimantadine, moroxydine, zanamivir and oseltamivir were less than 0.1%, which indicated the good specificity of the antibody. An ELISA was developed based on the antibody and applied to detect RBV in multi-food matrices. The sample preparation prior to detection only needed simple dilution after trichloroacetic acid extraction. The limits of detection were 1.07, 1.18 and 1.03 μg/kg in chicken muscle, eggs and duck muscle, respectively. Recoveries ranged from 89.0% to 112.7% with coefficients of variation below 13.0%. Ten blind samples of chicken muscle were analysed simultaneously by ELISA and liquid chromatography-tandem mass spectrometry, and a good correlation between the methods was observed. The results indicated that the high affinity antibody could be applied for the simple and fast detection of RBV in multi-food matrices.

Objectives: The objective of this study was to evaluate the effects of abacavir on intracellular ribavirin triphosphate and plasma ribavirin trough concentrations., Methods: Hepatitis C virus-infected subjects who had been cured or failed prior treatment were randomized to 8 weeks of ribavirin alone (N = 14; weight-based dosing) or weight-based ribavirin + abacavir (N = 14; 300 mg orally every 12 h). Ribavirin trough concentrations were measured on days 14, 28, 42 and 56; PBMCs for ribavirin triphosphate determination were sampled on days 28 and 56, pre-dose and at 6 and 12 h post-dose. ClinicalTrials.gov: NCT01052701., Results: Twenty-six subjects completed the study (24 males, 17 Caucasians, median age 52 years); 2 were excluded for missed pharmacokinetic visits. Fourteen subjects received ribavirin + abacavir and 12 received ribavirin alone. Mean ± SD plasma ribavirin trough concentrations (μg/mL) on days 14, 28, 42 and 56, respectively, were not significantly different with coadministration of abacavir (1.54 ± 0.60, 1.93 ± 0.54, 2.14 ± 0.73 and 2.54 ± 1.05) compared with ribavirin alone (1.48 ± 0.32, 2.08 ± 0.41, 2.32 ± 0.47 and 2.60 ± 0.62) (P > 0.40). Mean ribavirin triphosphate intracellular concentrations (pmol/10(6) cells) on days 28 and 56, respectively, did not differ statistically between abacavir users (11.98 ± 9.86 and 15.87 ± 12.52) and non-users (15.91 ± 15.58 and 15.93 ± 12.69) (P > 0.4). Adverse events were mild or moderate, except for three grade 3 occurrences of transaminitis, cholecystitis and low absolute neutrophil count that resolved and were judged not attributable to study medications., Conclusions: Abacavir did not significantly alter ribavirin or ribavirin triphosphate concentrations., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

A new analytical method for the determination of ribavirin in chicken muscle using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method and liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed and validated. Samples were extracted with acidified methanol (methanol:acetic acid, 99:1, v/v). The extract was further purified by QuEChERS method using primary-secondary amine (PSA) and C18. Finally, the extract was dried by nitrogen under 45°C and reconstituted in water. The separation was performed on a Hypercarb analytical column under a gradient elution. The mobile phase was composed of water buffered with ammonium acetate (2.0mM) and acetonitrile. The proposed method was validated according to the European Commission Decision 2002/657/EC. The values of the decision limit (CCα) and the detection capability (CCβ) were 1.1 and 1.5μg/kg, respectively. The mean recoveries of ribavirin ranged from 94.2% to 99.2%. The repeatability (expressed as coefficient of variation, CVr) of the method ranged from 4.5% to 4.9% and the reproducibility (CVR) of the method ranged from 4.8% to 5.4%. The method is demonstrated to be suitable for the determination of ribavirin in chicken muscle in conformity with the current EU performance requirements through validation. The total time required for the analysis of one sample, including sample preparation, was about 45min., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC-MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC-MS/MS. The method utilized a stable labeled internal standard (RBV-(13)C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.)

The current standard-of-care therapy in HCV consists in ribavirin (RBV) plus pegylated-interferon-α 2a or 2b and, for HCV-1 infected patients, also directly acting antivirals (DAAs). Despite the increase in the number of patients who reach sustained virological response (SVR) for HCV-1, a great inter-individual variability in the response to therapy remains. Whether new drugs are available in combination with RBV and Peg-IFN for HCV-1, the treatment of the other viral genotypes remains the same: this issue highlights the lasting importance of RBV and Peg-IFN in anti-HCV treatment. Moreover, a strong limiting factor to the usefulness of anti-HCV treatment remains the occurrence of adverse events, first of all hemolytic anemia, which have increased with the addition of DAAs, but is mainly an RBV-dependent effect. For these reasons, the monitoring of RBV exposure in the various compartments should be important. Since the routinely determination of RBV in the target cells as the hepatocytes is impracticable for of its invasiveness, the quantification in easier to obtain cells could be a good choice. In this work, we developed and validated an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay method to quantify RBV concentrations in peripheral blood mononucleated cells (PBMCs). QCs were prepared with RBV and RBV-monophosphate (RMP). Each sample was divided into two aliquots, which undergone the same extraction procedure: one was treated with acid phosphatase to convert RBV phosphorylated metabolites into free RBV, the other one was not-treated. The extracts were analyzed with reverse-phase column with UPLC-MS/MS. Calibration curves fitted a least squares model (weighed 1/X) for ribavirin levels in a range from 0.1 ng to 200 ng (mean r(2)=0.9993). Accuracy, intra-day and inter-day precision of the methods were in accordance with FDA guidelines. Moreover, phosphorylated QCs were used to assess the correct determination of total RBV concentration. We tested this method by monitoring RBV concentrations in PBMCs from 20 HCV+ patients, receiving alpha interferon-plus RBV combination therapy. This method showed to be reliable, precise, accurate and suitable for evaluation of intracellular RBV concentrations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An analytical method was developed for the determination of total residues of ribavirin and its phosphorylated metabolites in chicken and its products by hydrophilic interaction chromatography-tandem mass spectrometry. The samples were extracted with acetonitrile containing 1% (v/v) acetic acid under ultrasonic condition and then enzymatically hydrolysed with acid phosphatase at 37 degrees C. After liposoluble substances being removed with hexane, C18 and PSA dispersion solid phase extraction (DSPE) was introduced to cleanup procedures. The separation was performed on an ultra-performance hydrophilic interaction chromatographic (HILIC) amide column under a gradient elution using acetonitrile and 0.1% formic acid. The analytes were detected in the positive electrospray ionization and multi-reaction monitoring (MRM) mode. In the range of 1-200 microg/kg, the correlation coefficient was greater than 0.999. The limit of detection (LOD, S/N > or = 3) was 1 microg/kg and the limit of quantification (LOQ, S/N > or = 10) was 5 microg/kg. The recoveries of ribavirin spiked at three levels ranged from 67.8% to 112.7% with the relative standard deviations (RSDs) of 6.1%-13.6%. The results of the determination of ribavirin in various real samples showed that the method is simple, rapid, accurate and suitable for the determination of total residues of ribavirin and its metabolites in chicken and its products.

A method for the determination of ribavirin and amantadine in chicken has been developed by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS). After extracted by 1% (volume percentage) trichloroacetic acid solution-acetonitrile (1:1, v/v) and purified by a Supelco LC-SCX cartridge, the samples were loaded onto an Acquity UPLC BEH Hillic column (150 mm x 2.1 mm, 1.7 microm) and separated with gradient elution. The electrospray was operated in the positive mode and the samples were monitored by the multiple reaction monitoring (MRM) mode. The limits of quantification (LOQs, S/N = 10) of ribavirin and amantadine were 4.0 microg/kg. The calibration curves showed good linearity in the range of 10.0 - 100.0 microg/L, and the correlation coefficients (r(2)) were not lower than 0.99. When the spiked levels were 4.0, 8.0 and 20.0 microg/kg, the recoveries of ribavirin and amantadine in chicken ranged from 78% to 102.5%, with the relative standard deviations (RSDs) of 2.2% - 7.6%. The results indicate that the method is simple, rapid, sensitive and suitable for the qualitative and quantitative analyses of ribavirin and amantadine in chicken samples.

Background: The polar nucleoside drug ribavirin (RBV) combined with IFN-α is a front-line treatment for chronic hepatitis C virus infection. RBV acts as a prodrug and exerts its broad antiviral activity primarily through its active phosphorylated metabolite ribavirin 5´-triphosphate (RTP), and also possibly through ribavirin 5´-monophosphate (RMP). To study RBV transport, diffusion, metabolic clearance and its impact on drug-metabolizing enzymes, a LC-MS method is needed to simultaneously quantify RBV and its phosphorylated metabolites (RTP, ribavirin 5´-diphosphate and RMP). In a recombinant human UGT1A1 assay, the assay buffer components uridine and its phosphorylated derivatives are isobaric with RBV and its phosphorylated metabolites, leading to significant interference when analyzed by LC-MS with the nominal mass resolution mode., Results: Presented here is a LC-MS method employing LC coupled with full-scan high-resolution accurate MS analysis for the simultaneous quantitative determination of RBV, RMP, ribavirin 5´-diphosphate and RTP by differentiating RBV and its phosphorylated metabolites from uridine and its phosphorylated derivatives by accurate mass, thus avoiding interference., Conclusion: The developed LC-high-resolution accurate MS method allows for quantitation of RBV and its phosphorylated metabolites, eliminating the interferences from uridine and its phosphorylated derivatives in recombinant human UGT1A1 assays.

Objectives: To determine the relationship between erythrocyte and plasma ribavirin concentrations in hepatitis C virus (HCV)/HIV-coinfected patients, and to correlate ribavirin exposure with early and sustained virological response (EVR and SVR) and haemoglobin level reductions., Methods: Clinical and biological data from 68 HCV/HIV-coinfected patients were recorded at baseline, week 4 (W4), week 12 and at 24 weeks after completion of treatment. Plasma and erythrocyte ribavirin concentrations were determined 12 h after the final ribavirin dose (C(min))., Results: Erythrocyte ribavirin concentrations were 100-fold higher than plasma concentrations, with a significant relationship between them (P < 0.05). In patients with HCV genotype 1 or 4, a plasma ribavirin C(min) threshold of 1.95 mg/L at W4 tended to predict EVR [sensitivity 44%; specificity 87%; AUC 0.67 (95% CI 0.50-0.84)] and was predictive of SVR [sensitivity 58%; specificity 84%; AUC 0.71 (95% CI 0.51-0.90)]. Among patients with these HCV genotypes, an erythrocyte ribavirin C(min) threshold of 146 mg/L at W4 was found to be the best value for discriminating between responders and non-responders for both EVR [sensitivity 67%; specificity 75%; AUC 0.58 (95% CI 0.24-0.93)] and SVR [sensitivity 50%; specificity 80%; AUC 0.70 (95% CI 0.39-1.01)]. We also demonstrated a significant relationship between reduced haemoglobin levels and plasma ribavirin C(min) at W4 (P = 0.05)., Conclusions: Therapeutic drug monitoring may be useful for the management of anti-HCV treatment in HCV/HIV-coinfected patients.

A rapid and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of ribavirin (RBV) in rat brain was developed. Sample preparation required only two centrifuge steps before LC-MS/MS analysis and the chromatographic separation was achieved in isocratic conditions using an Atlantis T3 column with a nearly totally aqueous (95%) mobile phase. The method showed a good linearity over a concentration range of 5-1000ppb and satisfactory results in terms of accuracy., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Simple and sensitive spectrofluorometric and HPLC methods for the determination of ribavirin (RIB) were developed. The methods were based on the reaction of the 5'-hydroxyl group of the sugar moiety in RIB with dansyl chloride in a bicarbonate solution (pH 10.5) to form a fluorescent derivative. The first method was based on measuring the fluorescence intensity of the derivative in dichloromethane at 529 nm (excitation at 382 nm). The second method was HPLC separation of the derivative on a reversed-phase C(18) column with fluorescence detection. The linear ranges were 200-900 and 50-1000 ng/mL for the spectrofluorometric and HPLC methods, respectively. The derivatization product was characterized by spectroscopic methods. The proposed methods were successfully applied to analysis of the capsules.

Ribavirin pharmacokinetic and exposure effect trials based on either plasma or serum concentrations have yielded diverging results. This study aimed to compare ribavirin concentrations in serum and plasma and to investigate the influence of blood collection and preanalytical conditions on ribavirin concentration stability. Blood samples from patients with hepatitis virus C and receiving ribavirin were collected in plain (dry) tubes, in tubes containing ethylenediaminetetra-acetic acid or lithium-heparinate, in Type II Serum Separating Tubes with clot activator, or Type II lithium heparinate Plasma Separating Tubes. Different time and temperature conditions were tested before and after blood centrifugation. Ribavirin was determined using liquid chromatography-dual mass spectroscopy. Multiple-way analysis of variance was used for statistical analyses. Ribavirin concentrations showed a higher interlaboratory variability in serum than in plasma. Results were fairly stable over 2 hours in whole blood collected in dry or ethylenediaminetetra-acetic acid tubes and very stable up to 24 hours in serum or plasma kept in gel-containing tubes after immediate centrifugation. When Plasma Separating II gel tubes were kept at +4 degrees C or at ambient temperature for up to 24 hours before centrifugation, ribavirin concentrations decreased by 1% to 8% and 12% to 18%, respectively. These results suggest that blood samples should be collected in gel-containing tubes and centrifuged immediately, after which the tubes can be kept at ambient temperature for the next 24 hours. In case of clinical constraints, Plasma Separating II gel tubes can be kept at +4 degrees C for a maximum of 2 hours before centrifugation with limited impact on the measured concentrations.

A simple and accurate stability-indicating thin-layer chromatographic (TLC) method is developed and validated for the quantitative determination of ribavirin (RBV) in its bulk and with used for development consists of chloroform-methanol-acetic acid (60:15:15, v/v/v). The separated spots are visualized as bluish green spots after being sprayed with anisaldehyde reagent. RBV is subjected to different accelerated stress conditions. The drug is found to undergo degradation under all stress conditions, and the degradation products are well resolved from the pure drug with significantly different Rf values. The optical densities of the separated spots are found to be linear with the amount of RBV in the range of 5-40 microg/spot with a good correlation coefficient (r=0.9980). The limit of detection and limit of quantitation values are 1.40 and 4.67 microg/spot, respectively. Statistical analysis proves that the method is repeatable and accurate for the determination of RBV in the presence of its degradation products. The method meets the International Conference on Harmonisation/Food and Drug Administration regulatory requirements. The proposed TLC method is successfully applied for the determination of RBV, pure and in capsules, with good accuracy and precision; the label claim percentages are 98.8%+/-1.5%. The results obtained by the proposed TLC method are comparable with those obtained by the official method.

Ribavirin (RV) (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), is a synthetic purine nucleoside analog with a broad spectrum of antiviral activity. To better understand the mechanism of action of RV, as well as its pharmacokinetic characteristics, an assay that can allow specific, sensitive, and accurate measurement of RV in biologic samples is critical. In this way, diverse analytical methods have been established. In this work, we have recompiled these methods with the aim to present the different options for the RV determination.

Two simple, accurate, and reliable spectrophotometric methods have been developed for the determination of 2 antiviral drugs, acyclovir (ACV) and ribavirin (RBV), in their pharmaceutical formulations. These methods are based on oxidation of the 2 drugs with either cerium (IV) ammonium sulfate (Method A) or potassium persulfate (Method B). The products of oxidation in both methods are coupled with 3-methylbenzothiazolin 2-one hydrazone, producing a deep blue color with a maximum absorption wavelength at 630 nm. In Method A, the absorbance-concentration plots were linear over the ranges of 5-50 and 10-60 microg/mL with detection limits of 0.18 microg/mL (8 x 10(-7) M) and 0.63 microg/mL (2.58 x 10(-6) M) for ACV and RBV, respectively. In Method B, the ranges were 5-45 and 20-50 microg/mL with detection limits of 0.11 microg/mL (4.88 x 10(-7) M) and 1.40 microg/mL (5.73 x 10(-6) M) for the 2 drugs, respectively. The molar absorptivities were 4.1 x 10(3) and 3.65 x 10(3) L/mol/cm in Method A and 5.03 x 10(3) and 3.97 x 10(3) L/mol/cm in Method B for the 2 drugs, respectively. The proposed methods were applied successfully for the determination of the 2 drugs in their pharmaceutical formulations. The percentage recoveries +/- standard deviation were 99.57 +/- 0.86 and 100.82 +/- 0.46 for ACV; 99.41 +/- 1.08 and 100.35 +/- 1.03 for RBV. The results obtained were compared statistically with those given by official methods and showed no significant differences regarding accuracy and precision.

Eight spectrophotometric methods for determination of ribavirin have been developed and validated. These methods were based on the oxidation of the drug by different inorganic oxidants: ceric ammonium sulfate, potassium permanganate, ammonium molybdate, ammonium metavanidate, chromium trioxide, potassium dichromate, potassium iodate, and potassium periodate. The oxidation reactions were performed in perchloric acid medium for ceric ammonium sulfate and in sulfuric acid medium for the other reagents. With ceric ammonium sulfate and potassium permanganate, the concentration of ribavirin in its samples was determined by measuring the decrease in the absorption intensity of the colored reagents at 315 and 525 nm, respectively. With the other reagents, the concentration of ribavirin was determined by measuring the intensity of the developed colored reaction products at the wavelengths of maximum absorbance: 675, 780, 595, 595, 475, and 475 nm for reactions with ammonium molybdate, ammonium metavanidate, chromium trioxide, potassium dichromate, potassium iodate, and potassium periodate, respectively. Different variables affecting the reaction conditions were carefully studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9984-0.9998) were found between the absorbance readings and the concentrations of ribavirin in the range of 4-1400 microg/mL. The molar absorptivities were correlated with the oxidation potential of the oxidants used. The precision of the methods were satisfactory; the values of relative standard deviation did not exceed 1.64%. The proposed methods were successfully applied to the analysis of ribavirin in pure drug material and capsules with good accuracy and precision; the recovery values were 99.2-101.2 +/- 0.48-1.30%. The results obtained using the proposed spectrophotometric methods were comparable with those obtained with the official method stated in the United States Pharmacopeia.

A sensitive and specific method using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of total ribavirin in monkey liver is developed and validated. In this method, ribavirin and its phosphorylated metabolites are extracted with perchloric acid. The metabolites are converted to ribavirin using acid phosphatase and further purified using a NH2 solid-phase extraction (SPE) cartridge prior to LC-MS/MS analysis. [13C]Ribavirin is added with the extraction solution as an internal standard to obtain better accuracy and precision of the analysis. The MS/MS was selected to monitor 245-->113 and 250-->113 transitions using positive electrospray ionization for ribavirin and [(13)C]ribavirin. The calibration curve is linear over a concentration of 1.0-100 microg/g with a limit of quantitation (LOQ) of 1.0 microg/g. Mean inter-assay accuracy for QC at 1.0, 10 and 100 microg/g are 108, 99.7 and 99.7%, respectively. Mean inter-assay precision (CV) for QC at 1.0, 10 and 100 microg/g are 5.34, 5.24 and 4.59%, respectively. Extractability of total ribavirin from liver has been confirmed with liver obtained from monkey dosed with [14C]ribavirin. The method has been proven to be useful in the determination of total ribavirin concentration in liver from monkeys in mass balance study (10 mg/kg) and in 28 days toxicology study (300 mg/kg/day). It is also used to determine the total ribavirin concentration in human livers from hepatitis C patients received dose of 600 mg ribavirin twice daily.

A simple and sensitive fluorimetric method for determination of antiviral drugs: ribavirin, acyclovir, and amantadine hydrochloride has been developed. The method was based on the oxidation of these drugs by cerium(IV) in presence of perchloric acid and subsequent monitoring the fluorescence of the induced cerium(III) at lambdaexcitation 255 and lambdaemission 355 nm. Different variables affecting the reaction conditions such as the concentrations of cerium(IV), type and concentration of acid medium, reaction time, temperature, and the diluting solvents were carefully studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9978-0.9996) were found between the relative fluorescence intensity and the concentrations of the investigated drugs in the range of 50-1400 ng ml-1. The assay limits of detection and quantitation were 20-49, and 62-160 ng ml-1, respectively. The precision of the method was satisfactory; the values of relative standard deviations did not exceed 1.58%. No interference could be observed from the excipients commonly present in dosage forms. The proposed method was successfully applied to the analysis of the investigated drugs in pure and pharmaceutical dosage forms with good accuracy and precision; the recovery percentages ranged from 99.2 to 101.2+/-0.48-1.30%. The results obtained by the proposed fluorimetric method were comparable with those obtained by the official method stated in the United States Pharmacopoeia.

Accurate quantification of impurities existing as separate crystalline phases at trace levels in drug materials is an important issue in the pharmaceutical industry. In the present study, a thermoanalytical approach previously developed for quantifying trace levels of polymorphic impurity (form II metastable nuclei) in commercial salmeterol xinafoate powders has been successfully applied with slight modifications to ribavirin, an antiviral drug exhibiting roughly similar polymorph-dependent crystallization kinetics in melts to that of salmeterol xinafoate. Essentially, the approach involved modeling of the crystallization kinetics of both tested and reference drug materials in melts using the Avrami-Erofe'ev (AE) rate expression, derivation of a mathematical equation for relating the AE kinetic constant to the composition of reference polymorph mixtures, and the use of this derived equation (in the form of a calibration curve) to calculate the impurity contents of the tested samples from their computed AE constants. For ribavirin, modification of the latter equation by incorporation of an empirical exponent was found necessary to account for the composition-dependent changes in crystallization kinetics of the reference mixtures. Such modification has made possible the determination of polymorphic impurity content of as low as 0.004% (w/w) in ribavirin samples induced by different forms of grinding treatment.

The electrophoretic separation of ribavirin and 5-methylcytidine (internal standard) by capillary electrophoresis was examined. Separation was achieved using reverse polarity in a 100 mM borate electrolyte, pH 9.1, with 5 mM spermine added to reduce the electroosmotic flow. Sample preparation based on acetonitrile protein precipitation was found to be unsuitable for ribavirin analysis in patient samples due to insufficient sensitivity and interferences. Solid-phase extraction employing phenyl boronic acid cartridges provided cleaner separations. Using this approach with 500 microL sample and reconstitution of the dried extract into 100 microL of 33% v/v 100 mM phosphate buffer, pH 6.4 / 67% v/v acetonitrile, the detection and quantitation limits were determined to be 0.05 and 0.10 microg/mL, respectively, a sensitivity that is suitable for therapeutic drug monitoring of ribavirin in human plasma and serum samples. The method was validated and compared to a high-performance liquid chromatography (HPLC) method, showing excellent agreement between the two for a set of samples that stemmed from patients being treated with ribavirin and interferon-alpha-2b for a hepatitis C virus infection.

A sensitive, selective and reproducible HPLC method for determination of tiazofurin in rat brain was developed and validated. The method allowed determination and quantification of nanomolar concentrations of tiazofurin in brain and its regions (hippocampus, cortex and striatum) of treated animals. Separation of tiazofurin from other peaks from brain tissue was achieved by isocratic elution on reverse phase chromatographic column. The mobile phase consisted of 0.05 M sodium acetate pH 4.6. Run time was 15 min.

A rapid and sensitive HPLC-RP method for simultaneous determination of tiazofurin, its 5'-O acetyl and benzoyl esters and their active metabolite thiazole-4-carboxamide adenine dinucleotide was developed and validated. The method allowed determination and quantification of nanomolar quantities of these substances in cell extracts of treated cells, and was also used in kinetic studies of cellular uptake of tiazofurin and its esters from the cultivation medium. Separation of the analyzed substances from unidentified peaks from both biological materials was achieved by gradient elution, thus reducing the possibility of interference. The mobile phase consisted of a 0.1 M sodium-hydrogen phosphate, pH 5.1 and methanol. Run time was 22 min, with 5 min equilibration time.

Inosine 5 -monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme for the synthesis of GTP and dGTP. Two isoforms of IMPDH have been identified. IMPDH Type I is ubiquitous and predominantly present in normal cells, whereas IMPDH Type II is predominant in malignant cells. IMPDH plays an important role in the expression of cellular genes, such as p53, c-myc and Ki-ras. IMPDH activity is transformation and progression linked in cancer cells. IMPDH inhibitors, tiazofurin, selenazofurin, and benzamide riboside share similar mechanism of action and are metabolized to their respective NAD analogues to exert antitumor activity. Tiazofurin exhibits clinical responses in patients with acute myeloid leukemia and chronic myeloid leukemia in blast crisis. These responses relate to the level of the NAD analogue formed in the leukemic cells. Resistance to tiazofurin and related IMPDH inhibitors relate mainly to a decrease in NMN adenylyltransferase activity. IMPDH inhbitors induce apoptosis. IMPDH inhitors are valuable probes for examining biochemical functions of GTP as they selectively reduce guanylate concentration. Incomplete depletion of cellular GTP level seems to down-regulate G-protein function, thereby inhibit cell growth or induce apoptosis. Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes the dehydrogenation of IMP to XMP utilizing NAD as the proton acceptor. Studies have demonstrated that IMPDH is a rate-limiting step in the de novo synthesis of guanylates, including GTP and dGTP. The importance of IMPDH is central because dGTP is required for the DNA synthesis and GTP plays a major role not only for the cellular activity but also for cellular regulation. Two isoforms of IMPDH have been demonstrated. IMPDH Type I is ubiquitous and predominately present in normal cells, whereas the IMPDH Type II enzyme is predominant in malignant cells. Although guanylates could be salvaged from guanine by the enzyme hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), the level of circulating guanine is low in dividing cells and this route is probably insufficient to satisfy the needs of guanylates in the cells.

The ability of bacterial cultures to transform nucleosides to virazole, a riboside with antiviral activity, was studied. Qualitative and quantitative methods for estimation of the microbial activity in the biotransformation reaction were designed. A qualitative rapid method for analysis of a large number of cultures was developed. Thirty nine collection strains were tested for their capacity to bioconvert nucleosides to virazole. Intact cells grown in a liquid medium were used as a source of purine nucleoside phosphorylase. Purine nucleosides such as adenosine, inosine, guanosine and 30-percent ribose solution resulting from the riboxin hydrolysis were tested as the ribose donors. The best results were obtained with the ribose solution. No distinct correlation between the genus of the microorganisms and their capacity for bioconversion of certain substrates was observed. Several cultures which showed stable and high results with respect to all the tested substrates were detected.

Ongoing controversy regarding the hazards of exposure of healthcare workers to ribavirin aerosol led to the design and evaluation of a ribavirin aerosol evacuation system that scavenges the excess ribavirin. An oxyhood was placed within a croupette oxygen and cool mist tent. The tent was placed under negative pressure, using the hospital wall suction. Air samples were taken to determine ambient air concentrations and patient exposure concentrations and were compared to airborne ribavirin concentrations that occurred during administration of ribavirin without an evacuation system. The ribavirin evacuation system reduced ribavirin ambient air concentrations from 54 ug/m3 (mean) to 11 ug/m3 (mean) without reducing patient exposure concentrations. The results suggest that the system evaluated is an efficient and inexpensive means of reducing incidental employee exposure to ribavirin aerosol.

1. Two new exhaust control methods for scavenging and removing excess ribavirin aerosol were evaluated. Air samples were collected from pediatric intensive care rooms to determine the concentration of ribavirin. 2. Both control methods produced a 99.9% reduction between patient breathing zone concentrations and general room air concentrations. The average room air concentrations of ribavirin were 64 and 37 micrograms/m3 with the control methods in place, compared to average patient breathing zone concentrations of 45,000 and 58,000 micrograms/m3. 3. Additional control methods are reviewed, and published ribavirin environmental monitoring data is summarized. 4. Though the effect on fetal development in humans is not known, it is recommended that occupational exposure to ribavirin aerosol be minimized. A safe level for occupational exposure was calculated to be 91 micrograms/m3 for an 8-hour time weighted average.

To minimize the exposure of health care workers to aerosolized ribavirin, we designed a double tent containment system with circulating mist and suction applied between the tents and we evaluated the ability of this system to contain and evacuate aerosolized ribavirin. While the drug was administered with this system at three institutions, three air sampling pumps at different locations in the room and one worn by the investigator (personal breathing zone) collected air samples onto filters for assay of ambient ribavirin. Ribavirin determinations were made by high performance liquid chromatography. A 5- to greater than 20-fold decrease in ambient ribavirin concentrations was observed with the containment system compared with those detected with a single oxyhood in use. The personal breathing zone concentration was 566.0 micrograms/m3 with the oxyhood alone and fell to a mean concentration of 43.81 +/- 33.40 micrograms/m3 with the containment system. Though the risk to exposed health care workers is unknown, this system offers a simple way to decrease significantly occupational exposure to ribavirin.

Health care workers (HCW) are exposed to ribavirin aerosol during therapy of infants with respiratory syncytial virus infections. To assess the degree of HCW exposure, we analyzed air samples from patient rooms and HCW personal breathing zones during ribavirin aerosol delivery by ventilator (two samples), oxygen hood (two samples), and a new vacuum exhaust hood (four samples). HCW exposure to ribavirin during aerosol delivery by ventilator or vacuum exhaust hood system was substantially lower than HCW exposure during aerosol delivery by oxygen hood in rooms with adequate ventilation.

The anti-viral drug ribavirin primarily used for the treatment of respiratory syncytial virus (RSV) infection in infants is delivered by continuous nebulization with the Small Particle Aerosol Generator (SPAG). Clinical data suggest that the SPAG is an efficient nebulizer for ribavirin; it is also being used for the delivery of other pulmonary agents. The efficiency of a nebulizer can be defined in large part by delivering a major proportion of its output with a particle size of 1 to 5 microns which is small enough to penetrate the tracheobronchial tree. We assessed both the quality and quantity of the SPAG aerosol cloud with the aid of a multistage liquid impinger, utilizing 2% ribavirin and 1% cromolyn sodium solutions. The aerosol of either solution had a mass median aerodynamic diamter (MMAD) of approximately 1.2 microns, and this seemed insensitive to solution strength and nebulization period. Of the solutions of ribavirin and cromolyn sodium 92.3% and 95.0%, respectively, by weight of delivered particles, had aerodynamic diameters less than 5 microns and about 70-75% of the aerosol particles had an aerodynamic diameter in the 1-5 microns range. This implies that the aerosol cloud is adequate for delivery through a ventilator circuit. Performance was maintained over long (16 hours), continuous periods of delivery. The SPAG could be useful for delivery of other respiratory drugs by continuous nebulization.

A simple, sensitive, micromethod was developed to determine levels of ribavirin, or its active metabolic products, in human serum or urine. The procedure utilized the inhibition of measles virus cytopathic effect in BS-C-1 cells. Based upon maximum dilutions of human serum or urine containing ribavirin which inhibited the measles virus, the bioassay detected ribavirin in concentrations as low as 0.006 microgram/ml in serum or 0.03 microgram/ml in urine. Herpesvirus 1, parainfluenza virus 3 and reovirus 1 were also tested for sensitivity to ribavirin. Minimum inhibitory concentrations of ribavirin in serum or urine against these other viruses were no lower 0.32 microgram/ml.

The use of high-performance liquid chromatography (HPLC) to measure ribavirin in serum and other biological fluids has been limited by endogenous interfering substances. We report an HPLC procedure based on the extraction of ribavirin from serum, plasma, or cerebrospinal fluid with a boronate affinity gel, which uses a 3-methylcytidine internal standard. This assay is sensitive (to 0.4 microM), specific (no interference with 34 commonly prescribed drugs), reproducible (coefficients of variation from 5.4 to 22.4%), and linear (r = 0.999) over the range of clinically relevant concentrations in serum (from 0.5 to 50.0 microM). It also correlates well with the ribavirin radioimmunoassay (r = 0.992). This HPLC assay should be useful for measuring ribavirin in serum and other body fluids during clinical trials.

Reports that ribavirin was teratogenic in animals raised concerns of female health care personnel about possible occupational exposure during the care of infants having respiratory syncytial virus infections. Under simulated operational conditions, experiments were conducted to measure ribavirin residues in room air, in surface wipe samples, and in personal sampling devices worn by volunteers. There was exposure to a dispersible dust, presumably dried ribavirin, deposited inside the croupette or hood and on the bedding. Based on personal sampler data, it was estimated that, in a 12-h shift, the primary health care individual could inhale 2.4-9.1 micrograms ribavirin/kg bw.d. Recommendations to reduce the exposure of staff included the wearing of appropriate surgical gloves and a NIOSH-approved disposable respirator for dusts and mists while attending to the needs of the patients.

A combined gas chromatographic-mass spectrometric technique is described for the quantification of virazole in serum and urine. Proteins are removed by molecular filtration, lipids by extraction with dichloromethane and interfering endogenous constituents by acidic and basic ion-exchange resins. Virazole is quantified by monitoring the protonated molecular ions of the fully silylated derivatives of virazole (m/e 533) and the arabinose analog (internal standard) obtained by methane chemical ionization. The detection limit is 150 pg (0.6.10(-12) mole) of virazole injected. In serum 10 ng/ml (4.10(-8) mole) can be detected, 25 ng/ml quantified. In urine 0.5 microgram/ml can be quantified without preconcentration. Virazole was detected in serum for at least 96 h at the 70-ng/ml level.

Ribavirin, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxyamide (Virazole; Viratek, Inc., Covina, Calif.), has a broad spectrum of antiviral activity. However, the study of the absorption, metabolism, and excretion of this compound has been limited by the lack of an appropriate assay for ribavirin and its metabolites. Since ribavirin has definite potential for therapeutic use, we developed a radioimmunoassay to measure ribavirin levels in clinical specimens. To prepare an effective immunogen, ribavirin was monosuccinylated and coupled to ovalbumin. The competitive binding radioimmunoassay, in which tritium-labeled ribavirin and rabbit antiribavirin serum were used, was quantitative for ribavirin at concentrations of 1 pmol/100 microliter in urine or plasma samples. The rabbit antibody cross-reacted with the major metabolite of ribavirin, 1,2,4-triazole-3-carboxamide, at a low level (2 to 5%) which did not interfere with ribavirin binding until concentrations of 1,2,4-triazole-3-carboxamide 10- to 100-fold higher than ribavirin were present in mock samples, a condition not present in biological specimens. We used the ribavirin radioimmunoassay to determine the ribavirin concentration in mouse plasma after intraperitoneal administration, in the sera of adults from Sierra Leone after oral or intravenous administration for treatment of suspected Lassa fever, and in the sera of children in the United States after small-particle aerosol administration. Our experience with the radioimmunoassay indicates that it is sensitive, accurate, and reproducible. The assay will permit studies leading to a better understanding of the pharmacology and pharmacokinetics of this potentially useful antiviral drug.

Fifteen to 20 mg/ml ribavirin administered as a small particle aerosol for 10-18 h per day is currently the regimen generally used to treat experimental or naturally-occurring respiratory syncytial (RS) or influenza virus infections in humans. To determine if such prolonged treatment schedules could be reduced, cotton rats and mice were inoculated with RS or influenza B virus, respectively, and then treated with different concentrations of ribavirin small particle aerosols. Aerosols generated from reservoirs containing 60 mg/ml ribavirin given 2 h twice daily, protected cotton rats from RS virus and mice from influenza B virus as well as aerosols generated from reservoirs containing 20 mg/ml ribavirin given 11 h daily. Aerosols generated from reservoirs containing 40 or 20 mg/ml given 2 h daily were less efficacious. There was no evidence of intolerance or pulmonary histopathology in infected or uninfected animals exposed to any of the doses of ribavirin tested. These studies indicate that use of aerosols containing higher concentrations of ribavirin than generally used to treat respiratory virus diseases may permit significantly shorter treatment schedules without loss of efficacy or increase in toxicity.