Volume 4 • Issue 3 • 1000187 Mod Chem appl, an open access journal ISSN: 2329-6798 M o d e r n C h e m i s t r y & A p p l i c a t i o n s ISSN: 2329-6798 Modern Chemistry & Applications Khan et al., Mod Chem appl 2016, 4:3 DOI: 10.4172/2329-6798.1000187 Research Article Open Access Short and Robust HPLC-UV Method to Determine Serum Ribavirin Concentration without Evaporation Step Abdul Rafq Khan 1 *, Ali Al-Othaim 1 , Khalid Muhammed Khan 2 , Shazia Mrtaza 1 , Sara Altraif 1 , Waleed Tamimi 1 , Waqas Jamil 3 and Ibrahim Altraif 1 1 National Guard Health Affairs, Department of Pathology and Laboratory Medicine, Biochemical Metabolic Laboratory, Riyadh, Kingdom of Saudi Arabia 2 Haji Ibrahim Jamal Research Institute of Chemistry, University of Karachi, Karachi, Pakistan 3 University of Jamshoro, Jamshoro, Sindh, Pakistan Abstract 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 coeffcient 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 effciency 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 fow 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. *Corresponding author: Abdul Rafq Khan, National Guard Health Affairs, Department of Pathology and Laboratory Medicine, Biochemical Metabolic Laboratory, Riyadh, Kingdom of Saudi Arabia, Tel: +966505351680; E-mail: khanab@ngha.med.sa (or) abdulrafq_khan@yahoo.com Received August 15, 2016; Accepted August 23, 2016; Published August 30, 2016 Citation: Khan AR, Al-Othaim A, Khan KM, Mrtaza S, Altraif S, et al. (2016) Short and Robust HPLC-UV Method to Determine Serum Ribavirin Concentration without Evaporation Step. Mod Chem appl 4: 187. doi: 10.4172/2329-6798.1000187 Copyright: © 2016 Khan AR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Keywords: Ribavirin; HPLC; Analysis Introduction Ribavirin (RBV) is a guanosine ribonucleoside analog which has spectrum of antiviral activity against DNA and RNA viruses [1,2]. Chronic hepatitis C virus (HCV) is treated with α-2-interferon along with ribavirin analogs. Combination therapy has greatly improved the rates of biochemical and virological response compared to the patients treated with interferon alone [3-6]. Ribavirin absorbs quickly and maximum plasma concentration achieve within 1.5 hour afer oral administration. It slowly releases from kidney and gains steady state concentration afer 4 weeks. Ribavirin produces signifcant side efects like hemolytic anemia and varying biological responses. Terefore, monitoring of ribavirin concentration is important to reduce adverse efects, to drive dose modifcation and to optimize management of HCV-infected patients receiving combination treatment [7-9]. Analytical methods for the determinations of ribavirin reviewed in 2007 [10]. Ribavirin was analyzed by tandem mass spectrometry in monkey and rat brain [11,12]. Capillary electrophoresis and high performance liquid chromatographic methods with UV detection were used for the determination of ribavirin in human plasma [13,14]. A high performance liquid chromatographic method with solid phase extraction was also reported using phenylboronic acid cartridges for sample clean up followed by evaporation [15]. Extraction of RBV from plasma was performed using a novel method based on ultrafltration in one step that allows direct injection into the high-performance liquid chromatography without any prior steps of dryness or reconstitution [16]. Te ultrafltration technique is not commonly available in most of the lab and it limits the number of injection to 200-300 per column. Te HPLC column gives high pressure afer 200 samples with reduced resolution and bad peak shape. Afer reviewing most of the published methods for the determination of ribavirin in serum sample, we observed that all of these methods either involved the evaporation of purifed serum sample afer liquid - liquid extraction or afer solid phase extraction which consumed time and labor especially when ribavirin extracted with 3% formic acid. Afer loading serum sample on solid phase cartridges, the washing and elution process involved the gravity fow of solvents which did not work for several samples. Te third problem was direct injection of elution solvent into the HPLC column which gave a high solvent front merged with ribavirin peak. In this study we have developed a faster cleaning method of serum sample by using phenylboronic acid cartridges with vacuum elution of washing and extraction solvent. Te fnal elution solvent (3% formic acid) was directly injected into HPLC system without any evaporation step. Te vacuum manifold was used under low vacuum to make extraction process faster without any blockage of cartridges which was observed many times when solvent passed under gravity force from PBA cartridges. A precise and low fow rate was used on 3 μ, 3 mm × 150 mm C 18 column which completely resolved ribavarin peak with solvent front which was not possible with many published HPLC columns, mobile