Development and Validation of Liquid Chromatography–Tandem Mass Spectrometry for Simultaneous Determination of the Four Active Components of Danhong Freeze‐dried Powder Injection in Human Plasma

Abstract

A liquid chromatography‐tandem mass spectrometry was developed and validated for the simultaneous determination of protocatechuic aldehyde, danshensu, salvianolic acid B, and hydroxysafflor yellow A, with rutin as internal standard. Electrospray ionization patterns and efficiency of the analytes, along with their fragmentation, were investigated to achieve sensitive electrospray tandem mass spectrometry (ESI‐MS/MS) detection. Plasma samples were extracted by solid‐phase extraction columns, and the analytes were separated on a Dikma Diamonsil C₁₈ (200 mm×4.6 mm i.d., 5 µm) column using a mobile phase comprised of methanol:acetonitrile: 0.5% formic acid (20∶25∶55, v/v/v) at a flow rate of 1 ml/min. Detection was performed on a Finnigan TSQ ripple quadrupole tandem mass spectrometer in negative ion‐selected monitoring mode using electrospray ionization. Good linearity over the range 10–1000 ng/ml for danshensu, salvianolic acid B, and hydroxysafflor yellow A, and 5–500 ng/ml for protocatechuic aldehyde with acceptable accuracy and precision, respectively. All the validation data, such as accuracy, precision, and stability, were within the required limits. It was a potential platform for the pharmacokinetic and absorption, distribution, metabolism, and excretion (ADME) study of multiple constituent traditional Chinese medicine.

Keywords:

• Protocatechuic aldehyde
• Danshensu
• salvianolic acid B
• hydroxysafflor yellow A
• liquid chromatography‐tandem mass spectrometry
• quantitation