Determination of Colchicine in Blood by UPLC-Q/Orbitrap HRMS
As a potent alkaloid,colchicine has attracted much attention due to its unique structure and pharmacological properties.Colchicine has many medical applications,not only for the prevention and treatment of gouty arthritis,but also for the treatment of tumors,hepatitis and so on.Studies indicated that colchicine had low blood concentrations,so the method of determination of colchicine in blood should be more sensitive.Therefore,the objective of this article is to establish a sensitive method for analysis of colchicine in blood by liquid chromatogram-high resolution mass spectrum(UPLC-Q/Orbitrap HRMS).The effects of different mobile phases and chromatographic columns on the separation of colchicine were investigated.The results showed that the instrument had higher response value and better separation efficiency when Accucore™ Phenyl hexyl analytical column(2.1 mm×100 mm×2.6 μm)was used with methanol-water system as mobile phase.The blood sample(1.0 mL)was extracted with acetonitrile(1.0 mL)to precipitate protein.Then,its well-treated supernatant was separated on a Accucore™ Phenyl hexyl analytical column(2.1 mm×100 mm × 2.6 μm)with gradient elution that was run through the mobile phases consisting of 5 mmol/L ammonium formate aqueous solution(mobile phase A)and methanol(mobile phase B).The analytes were analyzed with UPLC-Q/Orbitrap HRMS under the mode of positive-ion ESI in full MS/ddMS2.Step collision energies of 35 eV,60 eV and 85 eV were used.Colchicine was linear with its calibration curve among the range of 0.5 to 100 ng/mL(R2=0.998 5),which showed good recovery and inter-day precision less than 7.1％.The method established here is simple,reliable,sensitive and appropriate for determination of colchicine in blood.In addition,colchicine is unstable when exposed to sunlight in solution.Attention should be paid to avoiding sunlight during the experiment so as to make the analysis results more reliable.
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