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-C(18) 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).