Modern Chemistry & Applications

Abstract

Short and Robust HPLC-UV Method to Determine Serum Ribavirin Concentration without Evaporation Step

Abdul Rafiq Khan, Ali Al-Othaim, Khalid Muhammed Khan, Shazia Mrtaza, Sara Altraif, Waleed Tamimi, Waqas Jamil and Ibrahim Altraif

Objective: Measurement of ribavirin (RBV) is important for therapeutic drug monitoring in hepatitis C patient. A simple and fast high performance liquid chromatography (HPLC) method developed and validated to measure ribavirin concentration in serum samples without an evaporation step.

Design and method: About 500 μl serum sample, 50 μl internal standard and 20 mM ammonium acetate buffer (pH=8.5) were mixed for 30 seconds and centrifuged. The supernatant was transferred into preconditioned phenyl boronic acid cartridges for solid phase extraction. All cartridges were washed two times with 1 mL of 20 mM ammonium acetate buffer under vacuum not exceeding 10 psi. Ribavirin and internal standard were eluted with 300 μl of 3% formic acid. An aliquot of 100 μl was injected into HPLC system.

Results: The method was linear in the range of 0.1-8.0 mg/l with 0.05 mg/l as limit of detection. The correlation coefficient of method comparison was 0.975 with p value of 0.116 showing a good reproducibility of results. The mean accuracy was checked at three different concentrations and found to be between 107-110% for all three levels. The extraction efficiency was 65.5% for ribavirin in the range of 0.1-8.0 mg/l and 71.2% for internal standard at 50 mg/l. The intra assay precisions were determined at 0.5, 2.5, and 5.0 mg/l and % CV were found to be 2.2%, 5.0%, 4.5% respectively. The injection reproducibility at three levels was 5.5%, 6.1% and 3.3%. Removal of gravity flow and evaporation step made this method faster and easy for routine analysis of ribavirin samples.

Conclusion: The newly developed HPLC method was faster, accurate and sensitive. It applied for determining serum ribavirin level in hepatitis C patient in our hospital.