A rapid HPLC/ESI‐MS/MS method for quantitative analysis of isovalerylshikonin in rat plasma

A new, fast and sensitive high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI‐MS/MS) method was developed and validated for isovalerylshikonin in rat plasma using emodin as internal standard (IS). The analyte was extracted from rat plasma with ethyl acetate, after 10% HCl treatment and protein precipitated by methanol. The compound was separated on an Ultimate? XB‐C₁₈ analytical column using a mobile phase of methanol–10 mM ammonium acetate in water–acetonitrile containing 0.05% formic acid (45 : 10 : 45, v/v/v) with isogradient elution. The analyte was detected in negative ion mode using multiple‐reaction monitoring. The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries and stability were determined. LLOQ was 9 ng/mL for isovalerylshikonin. Correlation coefficient (r) value for the linear range of the analyte was greater than 0.99. The intra‐day and inter‐day precision and accuracy were better than 8.52%. The relative and absolute recovery was above 86% and no matrix effects were observed for isovalerylshikonin. This validated method provides a modern, rapid and robust procedure for the pharmacokinetic study of the two compounds in rats after intravenous administration to rats (n = 4). Copyright © 2009 John Wiley & Sons, Ltd.