高效液相色谱法同时测定全血中的原卟啉和锌原卟啉  

Simultaneous determination of protoporphyrin Ⅸ and protoporphyrin Ⅸ zinc (Ⅱ) in blood by high performance liquid chromatography

作　　者：胡佳偲 孙晨 张昊[1] 霍宗利[1] HU Jia-cai;SUN Chen;ZHANG Hao;HUO Zong-li(Jiangsu Provincial Center for Disease Control and Prevention,Jiangsu Nanjing 210009,China)

机构地区：[1]江苏省疾病预防控制中心,江苏南京210009

出　　处：《江苏预防医学》2022年第3期272-276,共5页Jiangsu Journal of Preventive Medicine

基　　金：江苏省卫生与健康医学科研项目(Y2018080)。

摘　　要：目的建立可同时测定全血中原卟啉和锌原卟啉的高效液相色谱-荧光检测法。方法优化流动相、色谱柱、样品处理方法等,确定最佳参数。采用荧光检测器扫描选取最佳激发和发射波长。以保留时间定性,外标法定量,绘制标准工作曲线。计算方法的检出限、定量限、准确度、精密度、重复性和重现性。结果经优化,取血样30μl,加入170μl纯净水稀释,经涡旋混合器混匀,加入1.0 ml甲醇,涡旋混匀沉淀蛋白,于涡旋振荡器上混匀5 min,超声提取20 min,15289×g离心5 min,吸取上清液,50μl上机测定。采用反相键合固定相色谱柱Thermo Acclaim^(TM) 120 C_(18)(250 mm×4.6 mm,5μm 120?)分离,50 mmol/L乙酸铵水溶液(pH=6.0)和甲醇等度洗脱,原卟啉和锌原卟啉最适的激发/发射波长分别为396 nm/625 nm、411 nm/581 nm。原卟啉和锌原卟啉在1.0~50μg/L浓度范围内线性均良好(r值均>0.999);检出限均为12μg/L,定量限均为40μg/L;以抗凝牛全血为基质,在低、中、高3个浓度进行加标,回收率为78.2%~85.6%,相对标准偏差(RSD)为1.3%~4.2%,日间RSD为1.3%~5.5%。对12份样本进行测定,原卟啉和锌原卟啉含量分别为75~154μg/L和305~373μg/L,均低于慢性铅中毒标准。结论成功建立简单、快速可同时测定全血中原卟啉和锌原卟啉的高效液相色谱-荧光检测法,能够用于日常抗凝全血中的原卟啉和锌原卟啉含量测定。Objective To establish a high-performance liquid chromatography(HPLC)with fluorescence detection method for the simultaneous determination of protoporphyrin Ⅸ and protoporphyrin Ⅸ zinc(Ⅱ) in whole blood. Methods The experiment conditions such as mobile phase, chromatographic column, and sample processing method were optimized to determine the best parameters. The fluorescence detector was used to scan and select the best excitation and emission wavelength. The retention time was used for qualitative analysis and the external standard method for quantitative analysis;the standard working curves were drawn. Limit of detection(LOD),limit of quantification(LOQ),accuracy, prescision, repeatability and reproducibility of the method were calculated. Results After optimization, 30 μl whole blood sample was diluted by adding 170 μl purified water, which was mixed well by vortex mixer. Then 1.0 ml methanol was added and subjected to vortexed mix to precipitate protein by mixing 5 min on vortex oscillator and 20 min ultrasonic extraction, followed by 5 min centrifugation by 15 289 ×g. The supernatant was collected and 50 μl was injected for determination. The separation was performed on a Thermo Acclaim^(TM) 120 C_(18) column(250 mm×4.6 mm, 5 μm 120 A) with reversed-phase bonding. Isocratic elution was conducted with 50 mmoL/L ammonium acetate(pH6.0) and methanol buffer. The optimal excitation/emission wavelengths of protoporphyrin Ⅸ and protoporphyrin Ⅸ zinc(Ⅱ) were 396 nm/625 nm and 411 nm/581 nm, respectively. Both protoporphyrin Ⅸ and protoporphyrin(Ⅸ) zinc(Ⅱ) showed good linearity in the concentration ranges of 1.0-50 μg/L(all r>0.999),The LODs and LOQs were 12 μg/L and 40 μg/L for both protoporphyrin Ⅸ and protoporphyrin Ⅸ zinc(Ⅱ). Using anticoagulant bovine whole blood as matrix, the average spiked recovery rates at low, medium and high concentrations were 78.2%-85.6% and the relative standard deviations were 1.3%-4.2%(n=7),the relative standard deviations between days(RSDs) were 1.3%

关 键 词：高效液相色谱法 抗凝全血 原卟啉 锌原卟啉 

分 类 号：R113[医药卫生—公共卫生与预防医学]