高效液相色谱法测定心肌肽中组胺含量的可行性分析

Feasibility analysis of HPLC detecting histamine content in cardiomyopeptide

关键词:  降压物质  心肌肽  组胺  高效液相色谱法  丹磺酰氯柱前衍生法

Key words:  depressor substance  cardiomyopeptide  histamine  HPLC  dansyl chloride pre-column derivatization

摘要:

目的　建立和验证高效液相色谱（HPLC）测定心肌肽中组胺含量的方法。方法　采用多道生理记录仪记录受试样品致猫血压下降情况。用丹磺酰氯柱前衍生化高效液相色谱法测定心肌肽中组胺含量，心肌肽溶液经三氯乙酸沉淀后，取上清液以丹磺酰氯丙酮溶液为衍生化试剂，衍生化后进行高效液相色谱测定。色谱条件为：色谱柱C18柱(250 mm×4.6 mm，i.d.,5 μm)，以水－乙腈为流动相梯度洗脱，检测波长254 nm，并对此方法进行方法学考察（标准曲线的绘制、加标、回收率、精密度重复性、专属性）。分析样品中组胺含量与致猫血压下降值之间的相关性。结果　1~5号样品d_T/d_{S0.996，检出限为0.03 μg/mL，平均加标回收率>91.1%。日内与日间精密度RSD均<1.0%,重复性及专属性均符合生物样品分析要求。SPSS软件分析心肌肽中组胺的含量与降压物质检查法中降压反应比值（dT/dS）成良好的线性相关（r=0.974，P=0.036）。结论　采用HPLC法测定心肌肽溶液中组胺的含量，并结合降压反应比值(dT/dS)设定组胺含量的限值可能成为控制心肌肽原料药质量的良好方法。}

Abstract:

Objective　To establish and validate a method to determine histamine content in Cardiomyopeptide. Methods　HPLC procedures employing derivatization reagents, dansylchloride were applied for determination of histamine in cardiomyopeptide. the supernatant derivatisation with dansylchloride was performed after cardiomyopeptide solution precipitated by Trichloroacetic acid(TCA)，the histamine was determined by a gradient elution HPLC method on a C18 column (250 mm×4.6 mm, 5 μm) with a mobile phase consisting of acetonitrile and water and detection at 254 nm. The method was validated in terms of linearity, precision，accuracy and recovery. Drop of blood pressure in cats caused by cardiomyopeptide was meassued with BIOPAC MP100 system. Results　The method showed excellent linearity in the range 0.1-10 μg/mL，and the resulting correlation coefficients for histamine in the calibration range were 0.996，a limit of detection (LOD) of 0.03 μg/mL could be achieved，mean recovery rate was more than 91.1%，statistical analysis showed a good linear relationship between histamine content and the ratio of blood pressure dropping（d_T/d_S) （r=0.974，P=0.036）. Conclusion　The HPLC method was appropriate for the determination of histamine content in Cardiomyopeptide, which suggested the method could be a suitable alternative to conventional method in vivo experiments.