OBJECTIVE: To establish HPLC-quadrupole/electrostatic field orbitrap high resolution mass spectrometry for rapid determination of aconitine, mesaconitine, hypaconitine, benzoylaconitine, benzoylmesaconine and benzoylhypacoitine in rat plasma. METHODS: Internal standard lappaconitine was added into plasma sample, and methanol precipitated protein was used for pretreatment. HPLC-quadrupole/electrostatic field orbitrap high resolution mass spectrometry was adopted. HPLC condition was as follows as Sinochrom ODS-BP C18 column, mobile phase consisted of acetonitrile-1% formic acid solution (50 ∶ 50,V/V), the flow rate of 0.6 mL/min, sample size of 10 μL, column temperature of 25 ℃, automatic sampler temperature of 4 ℃. Mass spectrum scanning mode was full ion monitoring model, positive ion acquisition, mass charge ratios (m/z) of ion were 646.32(aconitine), 632.30(mesaconitine),616.31(hypaconitine),604.31(benzoylaconitine),590.29 (benzoylmesaconine),574.30(benzoylhypacoitine), 585.31 (internal standard). Six male Wistar rats were collected and given single dose of total alkaloid extract of Aconitum carmichaeli (4 mg/kg)intragastrically. Blood samples were collected before medication (0 h) and 0.5, 0.75, 1.25, 1.5, 2, 4, 6, 8, 10, 24 h after medication. Plasma concentration was determined and pharmacokinetic parameters were calculated by using PK-Solver V2.0 software. RESULTS:The linear range of 6 kinds of aconitum alkaloids in plasma were 0.1-10 μg/L (r>0.992). The limit of quantitation was 0.1 μg/L. Average recovery was higher than 75%, RSDs of intra-day and inter-day, matrix effects, stability test were all lower than 15%. The tmax of 6 kinds of aconite alkaloids were about 1.2 h; t1/2 were about 10 h; cmax of monoestertype aconite alkaloids were higher than those of diester-type aconite alkaloids. CONCLUSIONS: Established HPLC-quadrupole/electrostatic field orbitrap high resolution mass spectrometry is accurate, sensitive, simple and rapid, and can be used for plasma concentration monitoring of 6 kinds of aconitum alkaloids.
关键词
高效液相色谱法四极杆/静电场轨道阱高分辨率质谱法附子生物碱血药浓度大鼠
Keywords
HPLCQuadrurpole/electrostatic field orbitrap mass spectrometryAconitum alkaloidsPlasma concentrationRats