| PURPOSE:A valid,specific and sensitive method was adopted for quantitation of berberine hydrochloride in dog plasma using HPLC-MS/MS,then the method was validated.The method was successfully applied to determine the concentration of berberine after oral administration and intravenous administration.Then the assay was applied to perform the pharmacokinetics study of berberine hydrochloride in beagle dog plasma.METHOD:The method of liquid-liquid extraction was developed to prepare the dog berberine plasma samples,the simultaneous quantitative analytical assay was developed by LC-MS/MS.ZORBAX 300SB-C18 column(150 mm ×4.6 mm i.d.,5.0 μn)with the mobile phase of acetonitrile and water with 0.5%fomic acid at a flow rate of 1.0 mL/min.Positive electrospray ionization mass spectrometry with multiple reaction monitoring mode was applied to achieve 0.01-3 ng/mL linear range in dog plasma.Multiple reaction monitoring(MRM)was used for detection.Transition channels of the protonated molecular ions were m/z 336.1→ m/z 320.1,and m/z 256.3→ m/z 167.1 for berberine and benadrly.The specificity,precision,stability,linearity,the low limit of quantification,accuracy,recovery and matrix’ effects were evaluated for the validations of method for plasm according to relevant guidelines.The concentration of berberine in beagle dog plasma after oral administration at three dosage levels,multiple dosage level and intravenous injection at a single dosage level,were measured,respectively.The concentration-time profiles of berberine were drawn and pharmacokinetics parameters were caculated and analyzed using DAS 3.0 and SPSS 17.0.RESULTS:A valid,fast,sensitive and selective LC/MS/MS method was adopted for pharmacokinetics study of berberine hydrochloride in dog plasma.The method was fully validated and can be used to determine berberine in dog plasma.These assays,which were corresponded to the relevant norm,were suited for the study of preclinical pharmacokinetics of berberine after single and multiple doses in beagle dogs.After oral administration at three dosage levels of berberine hydrochloride to dogs,the pharmacokinetics parameters were caculated.Cmax was 1.1810.36 ng/mL,1.17±1.02 ng/mL,1.90±0.91 ng/mL,respectively;tmax.was 7.50±0.55h,7.17±0.98 h,9.33± 1.21 h,respectively;t1/2 was 20.42±7.07 h,17.50±15.62 h,24.85± 16.33 h,respectively;AUC0→t was 17.8517.40 ng/mL-h,32.26±27.39 ng/mL-h,33.10±13.85 ng/mL·h,respectively;AUCO0→∞ was 18.59±7.17 ng/mL-h,32.94±27.49 ng/mL·h,35.99±11.66 ng/mL·h,respectively;MRT was 24.29±2.90 h,28.21 ±5.58 h,28.26±2.17 h,respectively;CL/F was 620.22±263.69 L/h/kg,857.07±402.94 L/h/kg,1252.85±548.37 L/h/kg,respectively;Vd/F was 19249.53±10841.92 L/kg,23709.10117353.20 L/kg,48986.39±41143.18L/kg.After intravenous injection at a single dosage level of berberine hydrochloride to dogs,the pharmacokinetics parameters were caculated.Cmax was 283.53± 115.65 ng/mL,t1/2 was19.19±5.18h,MRT was 4.53±0.61h,5AU0→t was 78.06±7.54ng/mL·h,AUC0→∞was 109.07±23.30 ng/mL·h,Vd/F was 301.26±176.79 L/kg,CL/F was 9.53±2.05 L/h/kg.After oral administration at multiple dosage level of berberine hydrochloride to dogs,the pharmacokinetics parameters were caculated.Cmax was 3.01 ± 1.26 ng/mL;tmax was 9±0.89 h,t1/2 was 21.49 ±5.08 h,AUC0→t was 32.9618.75 ng/mL·h,AUC0→∞was 34.92±9.82 ng/mL·h,MRT was 28.7213.57 h,CL/F was 622.97±221.95 L/h/kg,Vd/F was 19390.80 ±9616.67 L/kg.The pharmacokinetic parameters were calculated using DAS 3.0 software and analyzed with SPSS 17.0.The result shows that the pharmacokinetic process of berberine hydrochloride in beagle dogs was non-linear kinetics.The absolute bioavailability of berberine in dog was caculated to be 2.29%by comparing the AUCO0→t 4caculated after oral administration of 10 mg/kg of berberine to that caculated after intravenous administration of the 1 mg/kg of berberine hydrochloride.After oral administration at multiple dosage level,the accumulation factor of berberine was 1.0,which showed that there was no accumulation in beagle dog plasma. |
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