Liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of cantharidin in biological specimens and application to postmortem interval estimation in cantharidin poisoning

Abstract

A rapid, sensitive liquid chromatography-tandem mass spectrometry (Lc-MS/MS) method was developed and validated for the determination and quantification of cantharidin in rats liver and kidney. After grinding with methanol, the supernatant was determined by Lc-MS/MS using an thermo Accucore C18 column (100 mm×2.1 mm, 2.6 μm) with a gradient elution of 0.1% formic acid and 0.1% acetonitrile, and in the subsequent analysis using selected reaction monitoring mode, three ion transitions were monitored for analyte. The limit of detection (LOD) was 0.741 ng/ml and the limit of quantitation (LOQ) was 2.471 ng/ml. Good linearity (R 2 = 0.9998) was observed for the analyte over the linear range (5-400 ng/ml). The LC-MS/MS method was applied to the analysis of rats liver and kidney in different postmortem intervals (6 h, 12 h, 24 h, 48 h, 72 h and 168 h after death) after a single dose (4 mg/kg) of cantharidin administration by gavage. At 72 h after death, the cantharidin concentration in livers and kidneys were significantly higher than that in other postmortem intervals. Linear regression equations between postmortem interval and lg postmortem cantharidin concentration in rats liver and kidney were Y = 0.007455*X + 1.332(R 2 = 0.863) and Y = 0.002689*X + 1.433 (R 2 = 0.115) respectively. the animal experiment demonstrated Lc-MS/MS method can be used to determine the postmortem cantharidin concentration in rats liver and kidney and the determination of cantharidin in the rats liver after death has potential value for postmortem interval estimation in cantharidin poisoning. Cantharidin, as the principal active ingredient of traditional Chinese medicine mylabris, was firstly discovered and used as anticancer drug in China 1. What is more, cantharidin-based pharmaceutical preparations, such as Fufangbanmao caspsules and Aidi injection, are now widely used in clinical treatment for cancers with good therapeutic efficacy 2-4. However, previous studies on the clinical efficacy and safety of cantharidin indicated that cantharidin had risk of hematotoxicity, gastrointestinal toxicity, liver or renal injury, neurotoxicity, cardiotoxicity and so on 5-7. Furthermore, many village doctors use the unprocessed mylabris for treatment in patients and the effects of different processing to pharmacodynamic action of canthardin were different 8,9. All of these lead to the lack of unified evaluation criteria for clinical efficacy and safety. As we know, the toxic dosage of cantharidin was similar to therapeutic dosage 10 and the potential risk of cantharidin poisoning far outweighs any potential benefit of therapeutic efficacy was caused by the unreasonable use or inappropriate medication in patients. Evenmore, cantharidin poisoning can lead to muti-organ failure even death 8 and the following conditions of cantharidin poisoning were also found in forensic identification center 8,11-14 such as patients who died after ingestion of cantharidin as aphrodisiac, ingested blister beetles accidentally, suicide by ingesting mylabris, homicide by poi...

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