Comprehensive Screening of Multiple Prohibited Substances in Chinese Traditional Patent Medicine by UHPLC-Q-Orbitrap HRMS.

Abstract

An ultrahigh performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) system was established for the rapid screening of doping agents in Chinese traditional patent medicine (CTPM), aiming to enhance the prevention and control of doping risks associated with herbal medicines. Samples were extracted by ultrasonic extraction with acetonitrile, while oily CTPM samples were extracted with 80% acetonitrile in water and purified using a Captiva EMR General Pigmented Dry cartridge. The extraction was concentrated under nitrogen flow, and the residues were dissolved, filtered, and detected using a Thermo Scientific UHPLC-Q-Orbitrap HRMS system. Separation was performed on an Agilent Zorbax Eclipse C18 column at 40°C with an injection volume of 5 μL and a gradient elution of 10-mM ammonium formate solution (pH 3.6) and acetonitrile as the mobile phase. The subsequent analysis was conducted using dual electrospray ionization in the Full MS/data-dependent secondary mass spectrometry scan mode. The method covers a total of 303 substances from 12 categories. Over 95% of the targets had limits of detection at or below 50 ng·g^-1 or ng·mL^-1 in CTPM. The method was validated for qualitative identification, including assessments of specificity, sensitivity, robustness, extraction recovery, matrix effect, and precision. The applicability of the method was demonstrated by the successful detection of higenamine (54%), ephedrine (42%), strychnine (11%), and morphine (2%) in 100 authentic samples. This paper presents a method for the rapid screening of doping agents in CTPM with high resolution, accuracy, and retrospectivity, reducing the risks of herbal medicine-induced doping violations.