Table 1.

Description of the three analytical methods developed for the analysis of ribavirin

	Method 1	Method 2	Method 3
Volume sample	200 μL	5 μL	500 μL
Extraction procedure	Addition of 700 μL of an ammonium acetate solution at pH 8.5 and 50 μL of a 10 mg/L solution of 6-methylaminopurine 9-ribofuranoside (IS) in water. Solid-phase extraction on a PBA cartridge equilibrated with methanol–formic acid (97:3 by volume) and 0.25M ammonium acetate. Eluction with 3mL of methanol–formic acid (97:3 by volume).	Dilution with 15 μL of water. Addition of 200 μL of a 1 μg/L solution of 3-methyl cytidine methosulfate (IS) in acetonitrile-water-perchloric acid (97:2:1 by volume). Vortex-mixing of the mixture for 30 seconds and centrifugation at 20,000 g for 10 minutes.	Addition of 500 μL of an ammonium acetate solution at pH 9 and 30 μL of a 100 mg/L solution of 3-methyl cytidine methosulfate (IS) in water. Equilibration of the PBA cartridge with methanol–formic acid (97:3 by volume) and 0.25 M ammonium acetate it pH 9. Eluction with 3 mL of methonal–formic acid (97:3 by volume).
Column	Atlantis HILIC Silica, 5 μm (150 × 2.1 mm) (Waters)	Luna HILIC, 3 μm (100 × 2.0 mm) (Phenomenex)	Uptisphere HDO-QS, 3 μm (150 × 4.6mm) (Interchim)
Mobile phase	Aqueous ammonium formate and acetonitrile	Aqueous ammonium acetate and acetonitrile	Aqueous orthophosphoric acid and acetonitrile
Detection mode	MS/MS, ESI, positive ionization mode. Ribavirin (ammonium adduct): m/z 262.1/113.2 (quantification); m/z 262.1/96.2, m/z 245.1/113.2 (confirmation); IS: m/z 282.2/150.1.	MS/MS, ESI, positive ionization mode. Ribavirin: m/z 245.2/113.1 (quantification); m/z 245.2/96.2 (confirmation); IS, m/z 258.1/126.1.	UV detection at 214 and 240 nm.
Analytical range	10–5000 ng/mL	100–7000 ng/mL	100–10000 ng/mL
Validation results	Intra-assay accuracy (n=5): CV<7.8%, MRE<6.5% Inter-assay accuracy (n=5): CV < 13.3 %, MRE < 5.7 % Recovery (n=3): > 92.8 %	Intra-assay accuracy (n=6): CV<3.9%, MRE < 3.1 % Inter-assay accuracy (n= 6): CV < 10.7 %, MRE < 6.7 % Recovery (n=3): > 92.9%	Intra-assay accuracy (n=6): CV <6.7 %, MRE <7.5 % Inter-assay accuracy (n=23): CV < 6.0 %, MRE < 1.8% Recovery (n= 5):> 55.3 %