溶血对液相色谱串联质谱检测血浆中儿茶酚胺及其代谢产物的影响

Influence of hemolysis on the results of plasma catecholamines and their metabolites by liquid chromatography-tandem mass spectrometry

目的探讨溶血对液相色谱串联质谱(LC-MS/MS)检测血浆儿茶酚胺[肾上腺素(E)、去甲肾上腺素(NE)、多巴胺(DA)]及其代谢产物[变肾上腺素(MN)、去甲变肾上腺素(NMN)、3-甲氧基酪胺(3-MT)]的影响。方法在正常血浆样本和轻度溶血[血红蛋白(Hb)为0.75 g/L]、中度溶血(Hb分别为1.50、3.00 g/L)、重度溶血(Hb为7.50 g/L)样本中分别加入儿茶酚胺及其代谢产物的标准品溶液,制成低值和高值样本。将上述样本采用弱阳离子固相萃取板进行前处理,每份样本重复检测3次。采用LC-MS/MS检测前处理提取液中的儿茶酚胺及其代谢产物的水平。结果无论MN、NMN、3-MT是高值还是低值,除3-MT在重度溶血(Hb为7.50 g/L)时未能被检测到外,其他不同溶血程度样本MN、NMN、3-MT检测结果与正常样本比较,差异均无统计学意义(P>0.05)。高值3-MT、MN、NMN 3次检测结果的变异系数(CV)均<15%。低值3-MT在中度溶血(Hb为1.50 g/L)时、低值NMN在重度溶血(Hb为7.50 g/L)时,3次检测结果CV均>15%。对于Hb为3.00 g/L的中度溶血样本,无论DA、E、NE是高值还是低值均无法检出;当Hb≤1.50 g/L时,E、NE和DA低值样本的检测结果与正常样本比较,差异无统计学意义(P>0.05);DA和NE高值样本的检测结果与正常样本比较,差异无统计学意义(P>0.05);而E高值样本的检测结果在轻度溶血(Hb为1.5 g/L)时明显降低(P<0.05)。高、低值DA、E、NE在Hb≤1.50 g/L时3次检测结果的CV均<15%。结论中、重度溶血对LC-MS/MS检测儿茶酚胺及其代谢产物有不同程度的影响。

Objective To study the influence of hemolysis on the results of plasma catecholamines[epinephrine(E),norepinephrine(NE)and dopamine(DA)]and their metabolites[metanephrine(MN),normetanephrine(NMN)and3-methoxytyramine(3-MT)]by liquid chromatography-tandem mass spectrometry(LC-MS/MS).Methods The standard solutions of catecholamines and their metabolites were added to normal plasma and plasma with different degrees of hemolysis[the levels of hemoglobin(Hb)were0.75g/L(mild hemolysis),1.50and3.00g/L(moderate hemolysis)and7.50g/L(severe hemolysis)to make the samples with low and high levels of catecholamines and their metabolites.All the samples were subjected to weak cationexchange solid phase extraction with3replicates.The catecholamines and their metabolites in extraction solutions were analyzed by LC-MS/MS.Results No matter the levels of MN,NMN,3-MT were high or low,the levels of MN,NMN and3-MT had no statistical significance among normal samples and all hemolytic samples(P>0.05),except the signal of3-MT could not be determined in severely hemolytic samples(the level of Hb was7.50g/L).The coefficients of variation(CV)of MN,NMN and3-MT in high-level samples from3replicates were<15%.When the levels of Hb were1.50g/L for low-level3-MT and7.50g/L for low-level NMN,the CV were>15%.When the level of Hb was3.00g/L,DA,E and NE were not determined in low-level or high-level samples.When the level of Hb was≤1.50g/L,the results of E,NE and DA in low-level samples and the results of DA and NE in high-level samples had no statistical significance among normal samples and hemolytic samples(P>0.05),but the result of E in high-level samples decreased,when the level of Hb was1.50g/L(P<0.05).The CV of E,NE and DA in lowlevel and high-level samples were all<15%,when the level of Hb was≤1.50g/L.Conclusions Moderate and severe hemolysis has influence on the results of catecholamines and their metabolites by LC-MS/MS.