Lou Anne McKown, Lori Payne lmckown@BASinc.com
BASi® Northwest Laboratory, McMinnville, OR
Ribavirin is an antiviral prodrug used for the treatment of Hepatitis C and respiratory syncytial virus (RSV) among other common viruses. A method for the analysis and quantification of ribavirin in rat plasma utilizing HPLC-MS/MS with updated HPLC column technology has been developed and validated according to the FDA guidance to the industry. The assay displays good linearity with correlation coefficient >0.99 over a range of 10 “ 5000 ng/mL using a 25 Î¼L sample precipitated with 5mM ammonium acetate in 95% acetonitrile in water after the addition of a stable label internal standard. The supernatant is analyzed by HPLC utilizing a HILIC column with detection by MS/MS incorporating a turbo-ionspray interface in positive mode. The choice of HILIC column was essential for adequate retention and acceptable peak shape of ribavirin within 2 minutes of total analysis time. This method greatly facilitated the high throughput analysis of preclinical samples.
The synthetic compound with the IUPAC name 1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3- carboxamide was first produced in 1972 and reported to exhibit broad spectrum activity against DNA and RNA viruses. The structure of ribavirin is shown in Figure 1 and the molecular weight is 244 with molecular formula of C8H12N4O5. The high abundance of heteroatoms in the structure of ribavirin and the number of atoms available for hydrogen bonding lends itself to be very polar and water soluble. These physical characteristics present a challenge for chromatographic resolution and the recent advances in column chemistries for HPLC separation provide ample new opportunities to develop a rapid method for the determination of ribavirin concentrations in biological samples. Numerous different column chemistries were evaluated and produced a new HPLC-MS/MS method for the rapid analysis of ribavirin. This method was validated to current GLP standards in rat plasma over a range of 10-5000 ng/mL and provided reliable results for the pharmacokinetic evaluation of preclinical samples.
 J. Med. Chem. 15 (1972) 1150.  Science 177 (1972) 705.
Figure 1. The molecular structure of ribavirin.
Representative chromatograms of Ribavirin from traditional C-18 HPLC columns:
|Figure 2. Chromatogram of Ribavirin from a Phenomenex Synergi 4 m Hydro-RP 80Ã… 2 x 50mm eluted isocratic with 5% ACN in 0.1% formic acid in water.||Figure 3. Chromatogram of Ribavirin from a GL sciences Inertsil ODS-EP 5 mm 2.1 x 33mm eluted isocratic with 5% ACN in 0.1% formic acid in water.||Figure 4. Chromatogram of Ribavirin from a Waters Xbridge Shield RP18 5 mm 2.1x 50 mm eluted isocratic with 5% ACN in 0.1% formic acid in water.|
Representative chromatograms of Ribavirin from underivatized silica columns:
|Figure 5. Chromatogram of Ribavirin from a Phenomenex Luna Hilic 3 mm 2 x 100 mm eluted isocratic with 95% ACN in water||Figure 6. Chromatogram of Ribavirin from a Phenomenex Luna Hilic 3 mm 2 x 100 mm eluted isocratic with 5 mM ammonium acetate in 95% ACN|
|Figure 7. Representative chromatograms of plasma extracts of ribavirin. Upper left: 10 ng/mL ribavirin (LOQ), lower left: 5000 ng/mL ribavirin (ULQ), upper and lower right are ribavirin-13C5 (internal standard).|
Figure 8. Example standard curve
Resolution and retention of ribavirin on the Phenomenex Luna Hilic column is very sensitive to changes in the constitution of injection solvent and injection volume so it dictated the treatment of the analyte in the preparation of samples. A simplified protein precipitation sample preparation follows:
Ribavirin and ribavirin-13C5 were purchase from Toronto Research Chemicals, Inc. Na2EDTA rat plasma was purchased from Biochemed, inc. Ammonium acetate was purchased from Mallinckrodt. Methanol and acetonitrile were manufactured by Burdick and Jackson, and the water was deionized and filtered (0.2 Î¼m) using a Milli-Q Plus® system. The HPLC-MS/MS system consisted of a Shimadzu liquid chromatography system including 2
isocratic pumps, autosampler, a pre-column filter with replaceable frit (0.5 Î¼m), Phenomenex Luna Hilic column (3 mm, 2.0 x 100 mm), and a 6 port valve connected to a API Sciex 365 mass spectrometer with Ionics Interface upgrade and Analyst instrument control software (Applied Biosystems, Foster City, CA). The mass spectrometer was run in positive mode with multiple reaction monitoring of 245 †’ 113 transition for ribavirin and 250 †’ 113 transition for the internal standard.
A new method for the quantification of ribavirin in rat plasma was successfully validated according to FDA guidance to the industry. Ribavirin was isolated from rat plasma by protein precipitation of 25 Î¼L of spiked plasma with 5 mM ammonium acetate in 95% acetonitrile in water (mobile phase) after the addition of 50 Î¼L of a solution of 500 ng/mL ribavirin-13C5 in mobile phase. The method utilized a Phenomenex Luna Hilic column for HPLC purification which was very sensitive to the many factors such as buffer concentration and injection volume which influence the chromatography of an analyte.
Precision (% CV) and accuracy (% bias) of the quality control samples at concentrations of 10, 30, 1000, and 3800 ng/mL were 4.6- 17.5 %CV and -6.4-3.3 % bias over 3 method performance batches (n=18). The accuracy and precision was within ±15% for validation samples independently subjected to the following conditions: 4 freezethaw cycles at -80°C, 24 h at room temperature (rt), 1 month of storage at -80°C, or extracts stored at rt for 4 days. No interference in the quantification of ribavirin was seen from 6 different lots of rat plasma and the method was not impacted by carryover of ribavirin or the internal standard in the system. This method was used to quantify ribavirin in preclinical samples.