Bioanalysis of Ribavirin in Rat Plasma by High Pressure Liquid Chromatography – Tandem Mass Spectrometry (HPLC-MS/MS)
Lou Anne McKown, Lori Payne
Bioanalytical Systems, Inc., 3138 NE Rivergate #301C, McMinnville, OR 97101
Abstract
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 µL sample precipitated with 5mM ammonium acetate in 95% acetonitrile in water after the addition of an isotopically labeled 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.
Introduction
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[1] and reported to exhibit broad spectrum activity against DNA and RNA viruses[2]. 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 makes it very polar and water soluble. These physical characteristics present a challenge for chromatographic resolution. Recent advances in column chemistries for HPLC separation provide new opportunities to develop a rapid method for the determination of ribavirin concentrations in biological samples. Numerous different column chemistries were evaluated, resulting in 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.
Figure 1. The molecular structure of ribavirin.
Materials and Methods
Ribavirin and ribavirin-13C5 were purchased 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 µm) 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 µm), Phenomenex Luna HILIC column (3 µm, 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.
Results and Discussion
The published methods for the HPLC-MS/MS analysis of ribavirin involve chromatographic separation with traditional C-18 HPLC columns. Suitable retention and/or peak shape was not obtained with published methods or with other common columns used for polar compounds. Representative chromatograms are presented in Figures 2-4.
Figure 2. Chromatogram of Ribavirin from a Phenomenex Synergi 4 µ Hydro-RP 80Å 2 x 50 mm 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 µm 2.1 x 33 mm eluted isocratically with 5% ACN in 0.1% formic acid in water.
Figure 4. Chromatogram of Ribavirin from a Waters Xbridge Shield RP18 5 µm 2.1x 50 mm eluted isocratically with 5% ACN in 0.1% formic acid in water.
A selection of underivatized silica columns were tested and suitable retention was achieved on a Phenomenex Luna HILIC column as seen in Figure 5.
Figure 5. Chromatogram of Ribavirin from a Phenomenex Luna HILIC 3 µm 2 x 100 mm eluted isocratically with 95% ACN in water
Modification of the mobile phase produced significant differences in the retention time and the peak shape of ribavirin on the Luna HILIC column. The factors which had the most significant impact on the chromatography were buffer concentration and injection volume. Total overall buffer concentration of 5 mM ammonium acetate in 95% ACN in water at 0.5 mL/min exhibited symmetrical peak shape and increased retention of ribavirin by 0.3 min compared to no buffer as illustrated in Figure 6. Increase in the buffer concentration resulted in greater retention of ribavirin but peak shape suffered greatly. Decrease in the buffer concentration resulted in less retention, although the peak shape remained symmetrical. Injection volumes in excess of 5 µL resulted in severe tailing and deteriorated peak shape.
Figure 6. Chromatogram of Ribavirin from a Phenomenex Luna HILIC 3 µm 2 x 100 mm eluted isocratic with 5 mM ammonium acetate in 95% ACN
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 dictating the treatment of the analyte in the preparation of samples. Small sample volume was also critical in this method as it targeted samples from a small species (rat). Protein precipitation as the mode of sample preparation ideally complimented the restraints of the desired method. After minimal fine tuning, the procedure to purify ribavirin from plasma involved adding 25 µL of plasma to a 96 well plate followed by 50 µL of a 500 ng/mL solution of ribavirin-13C5 in mobile phase and 0.5 mL mobile phase. The plate was centrifuged and 0.4 mL of the supernatant liquid was transferred to a clean 96 well plate and 5 µL was injected directly into the HPLC-MS/MS system. A divert valve was installed on the system in between the HPLC column and the MS interface and was set to divert the first 50 seconds of the effluent to waste to limit the exposure of the MS to the dissolved proteins. Representative chromatograms from the lower limit of quantification (LOQ) and the upper limit of quantification (ULQ) are shown in Figure 7.
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).
A standard curve was generated from 8 concentrations over a range of 10 -5000 ng/mL of ribavirin using a linear 1/x2 fit of the ratio of peak areas of ribavirin to the internal standard, resulting in r2 correlations of greater than 0.99. A representative standard curve can be found in Figure 8. Precision (% CV) and accuracy (% bias) of the quality control samples at concentrations of 10, 30, 1000, and 3800 ng/mL ranged from 4.6 to 17.5 %CV and from -6.4 to 3.3 % bias over 3 method performance batches (n=18). The accuracy and precision were within ±15% for validation samples independently subjected to the following conditions: 4 freeze-thaw 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.
Figure 8. Example standard curve
Conclusion
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 µL of spiked plasma with 5 mM ammonium acetate in 95% acetonitrile in water (mobile phase) after the addition of 50 µL of a solution of 500 ng/mL ribavirin-13C5 in mobile phase. The method utilized a Phenomenex Luna HILIC column for HPLC separation. This method was used to quantify ribavirin in preclinical samples.
References
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- R.W. Sidwell, J.H. Huffman, G.P. Khare, L.B. Allen, J.T. Witkowski, R.K.Robins, Science 177 (1972) 705.
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