title: Concentration Monitoring of Plasma Ribavirin: Validation of a Liquid Chromatography-Mass Spectrometric Method and Clinical Sample Collection.
abstract: BACKGROUND
  A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for routine measurement of ribavirin concentrations in EDTA-anticoagulated plasma.
  
  
  METHODS
  After protein precipitation, we used a bridged ethylene hybrid (hydrophilic interaction) chromatography column, 0.1 mmol/L ammonium formate pH 3.0, and a gradient of 85%-96% acetonitrile to achieve baseline separation of ribavirin from isobaric uridine. Quantitation was assured using both primary (m/z 245.3 > 113.0) and secondary transitions (m/z 245.3 > 96.0) of the protonated species. Chromatographic separation and column washing also negated interference from major phospholipid species.
  
  
  RESULTS
  There was a linear relationship between concentration and response to 10 mg/L, with a minimum detectable level and a minimum level of quantitation both of 0.1 mg/L. Imprecision within the assay was <10% at 0.1 mg/L and <6% between assays for concentrations >0.4 mg/L. Bias was <4%. In clinical samples (n = 12), there was no difference in ribavirin concentrations obtained by an established liquid chromatographic assay with ultraviolet detection. Ribavirin concentrations were stable in plasma stored at room temperature for 3 days but then decreased significantly on day 7. Plasma concentrations were stable for 15 weeks at -20 °C. Concentrations in plasma separated from whole blood at room temperature fell by a median of 19.4% at 4 hours and then rose substantially (median 251% by 3 days). Dose-normalized ribavirin concentrations reached a steady state after a mean of >6 weeks treatment in 76 patients with hepatitis C.
  
  
  CONCLUSIONS
  A hydrophilic interaction liquid chromatography-tandem mass spectrometric method to measure ribavirin in plasma was developed. Samples for ribavirin estimation should be kept at 4 °C, separated within 2 hours of collection and stored at 4 °C before analysis, with long-term storage at -20 °C. This method was applied to a study of the ribavirin therapeutic monitoring in patients with hepatitis C.
authors.affiliation: Institute of Liver Studies, Denmark Hill, King's College Hospital, London, United Kingdom.
authors.forename: Nigel W
authors.initials: NW
authors.lastname: Brown
authors.forename: Phillip E
authors.initials: PE
authors.lastname: Morgan
authors.forename: Kosh
authors.initials: K
authors.lastname: Agarwal
authors.forename: John M
authors.initials: JM
authors.lastname: Tredger
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000998:Antiviral Agents / Q000097:blood; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods*; D004355:Drug Stability; D004356:Drug Storage; D004492:Edetic Acid / Q000737:chemistry; D006526:Hepatitis C / Q000188:drug therapy; D006801:Humans; D008875:Middle Aged; D012254:Ribavirin / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors; D055815:Young Adult
chemical_list: D000998:Antiviral Agents; D012254:Ribavirin; D004492:Edetic Acid