Abstract: AIM: To develop sensetive and specific LC-MS/MS-based methods for measurement of plasma tanshinol (TSN), protocatechualdehyde (PCA), and salvianolic acid A, B, and D (SAA, SAB, and SAD)in supporting the pharmacokinetic evaluation of Cardiotonic pills, a standardized danshen-containing cardiovascular phytomedicine. METHODS: Electrospray ionization (ESI)patterns and efficiency of the analytes, along with their fragmentation, were investigated to achieve sensitive ESI-MS/MS detection.Sample preparation was also studied to effectively recover the analytes from plasma.Meanwhile, chromatographic conditions were optimized to avoid matrix component interference on analyte ESI-detection. The newly developed analytical methods were evaluated with respect to accuracy, precison, selectivity, sensitivity, and stability. RESULTS: The negative ion ESI gave a high ionization efficiency for the analytes.The precursor-to-product ion pairs m z 197→135, 136→108, 493→295, 717→519, and 417→197 were used for sensitive and specific SRM of TSN, PCA, SAA, SAB, and SAD, repectively.For the best measurement of the analytes, two methods were developed, i.e., one for determination of plasma TSN and the other one for simultanious determination of plasma PCA, SAA, SAB, and SAD.The sample preparation for the two quantification methoids involved acidifying the plasma sample with HCl before EtOAc-based liquid-liquid extraction;the extraction efficiency was 57.7%-61.6% for TSN from plasma or 38.0%-58.9% for PCA、SAA、SAB, and SAD in parallel from plasma.Matrix-matched standard curves of the peak area of a given analytes versus the nominal plasma concentration were linear for concentrations of TSN between 2.7 ng/mL and 2 000.0 ng/mL, with a correlation coefficient 0.999 and a lower limit of quantification 2.7 ng/mL, while linear dynamic ranges for PCA, SAA, SAB, and SAD were 1.37 or 4.1-1000.0 ng/mL, with correlation coefficients>0.99 and lower limits of quantification 1.4 ng/mL for PCA and 4.1 ng/mL for SAA, SAB, and SAD.Both the quantification methods demonstrated a within-run assay accuracy and presion 90%-112% and 2.5%-15%, respectively and a between-run data 91%-110% and 0.7%-9.4%, respectively.Further, the applicability of the newly developed analytical methods was demonstrated in a pilot plasma pharmacokinetic study in a beagle dog receiving an intravenous dose of injectable extract from Cardiotonic pills. CONCLUSION: Sensetive and specific LC-MS/MS-based methods for measurement of plasma TSN and for measurement of plasma PCA, SAA, SAB, and SAD in parallel are developed and validated, which are applicable to pharmacokinetic evaluation of Cardiotonic pills.

Key words: tanshinol, protocatechualdehyde, salvianolic acids, LC-MS/MS, plasma concentration