HPLC-FD同时测定人血浆中沙库巴曲缬沙坦3种成分的浓度及其应用

Simultaneous Determination of Three Components of Sacubitril/Valsartan in Human Plasma by HPLC-FD and Its Application

关键词:  沙库巴曲缬沙坦  LBQ-657  药动学  高效液相色谱-荧光检测法

Key words:  sacubitril/valsartan  LBQ-657  pharmacokinetics  HPLC-FD

摘要:

目的 建立人血浆样品中沙库巴曲、缬沙坦及其主要代谢产物LBQ-657的高效液相色谱-荧光检测(HPLC-FD)分析方法。方法 采用乙腈沉淀法处理血浆样品，SN-38作为内标进行内标法定量。采用Agilent Eclipse SB-C₁₈色谱柱(250 mm×4.6 mm，5 μm)，荧光检测器；以甲醇-磷酸二氢钠缓冲液(pH 3.0)(67∶33)为流动相；流速1.0 mL·min^–1；柱温25 ℃；进样量20 μL。采用多波长法，在0~6 min，λ_EX=380 nm，λ_EM=430 nm，定量检测SN-38；6~9.4 min，λ_EX=255 nm，λ_EM=314 nm，定量检测LBQ-657；9~12 min，λ_EX=255 nm，λ_EM=374 nm，定量检测缬沙坦；12~18 min，λ_EX=255 nm，λ_EM=314 nm，定量检测沙库巴曲。5例患者口服100 mg沙库巴曲缬沙坦钠片后1.5，3 h分别采样，检测血浆中沙库巴曲、缬沙坦及LBQ-657浓度。结果 人血浆样品中各成分在0.1~20 μg·mL^-1内线性关系良好(r>0.999)，低、中、高质控血浆样品的提取回收率为80.30%~96.45%。日内、日间精密度、准确度和稳定性均符合生物样品分析要求。结论 建立的HPLC-FD定量分析方法快速、灵敏、准确，可用于人血浆中沙库巴曲、缬沙坦及其主要代谢产物LBQ-657的含量测定。

Abstract:

OBJECTIVE To establish an analytical method for simultaneous determination of sacubitril, valsartan and its main metabolite LBQ-657 in human plasmas by high performance liquid chromatography-fluorescence detection(HPLC-FD). METHODS Precipitation method was used to treat plasma samples via acetonitrile and SN-38 applied as internal standard. Agilent Eclipse SB-C₁₈(250 mm×4.6 mm, 5 μm) was used. The chromatographic separations were accomplished using multi-wavelength with a mobile phase composed of methanol and sodium dihydrogen phosphate buffer(pH 3.0) at ratio of 67:33. The column temperature was maintained at 25℃ with the flow rate of 1.0 mL·min^-1.The injection volume was 20 μL. The multi-wavelength conditions were 0-6 min, λ_EX=380 nm, λ_EM=430 nm, for SN-38; 6-9.4 min, λ_EX=255 nm, λ_EM=314 nm, for sacubitril; 9-12 min, λ_EX=255 nm, λ_EM=374 nm, for valsartan; 12-18 min, λ_EX=255 nm, λ_EM=314 nm, for LBQ-657. Sacubitril and valsartan sodium tablets(dose 100 mg) was administered to 5 patients and sampling after 1.5, 3 h. HPLC-FD was used to detect concentration of sacubitril, valsartan and its main metabolite LBQ-657 in human plasma. RESULTS The calibration curves were linear over the range of 0.1-20 μg·mL^-1(r>0.999). The extraction recovery of sacubitril, valsartan and LBQ-657 were found from 80.30% to 96.45%. According to the intra- and inter-day precisions, accuracy and stability, demonstrating that the method was confirmed to be accurate, precise and stable. CONCLUSION The developed method is successfully applied for determination of sacubitril, valsartan and its main metabolite LBQ-657 in human plasma.