Tomšíková H, Solich P, Nováková L
Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic.
Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic.
J Pharm Biomed Anal. 2014 Jul;95:265-72. doi: 10.1016/j.jpba.2014.03.012. Epub 2014 Mar 15.
Elevated levels of pteridines can indicate the activation of cellular immune system by certain diseases. No work dealing with the simultaneous determination of urinary neopterin, biopterin and their reduced forms has been published. Therefore, a new SPE-UHPLC-FD method for the analysis of these compounds has been developed. The main emphasis was put on the stability of dihydroforms during the sample processing and storage. As a stabilizing agent, dithiothreitol, at various concentrations, and various pH values (3.8-9.8) of working solutions were tested. Chromatographic separation was performed under HILIC isocratic conditions on BEH Amide column. The method was linear for the calibration standard solutions in the range of 10-10,000 ng/ml (dihydroforms) and 0.5-1000 ng/ml (oxidized forms), and for real samples in the range of 25-1000 ng/ml (dihydroforms) and 1-100 ng/ml (oxidized forms). The development of a new SPE sample preparation method was carried out on different types of sorbents (based on a mixed-mode cation exchange, porous graphitic carbon and a polymer comprising hydrophilic and hydrophobic components). Final validation was performed on a MCAX SPE column. Method accuracy ranged from 76.9 to 121.9%. The intra- and inter-day precision did not exceed 10.7%. The method provided high sensitivity for the use in routine clinical measurements of urine (LLOQ 1 ng/ml for oxidized forms and 25 ng/ml for dihydroforms). Average concentrations of biopterin, neopterin, and dihydrobiopterin found in urine of healthy persons were related to the mol of creatinine (66.8, 142.3, and 257.3 μmol/mol of creatinine, respectively) which corresponded to the literature data. The concentration of dihydroneopterin obtained using our method was 98.8 μmol/mol of creatinine.
蝶啶水平升高可能表明某些疾病激活了细胞免疫系统。目前尚未有关于同时测定尿中新蝶呤、生物蝶呤及其还原形式的研究报道。因此，开发了一种用于分析这些化合物的新型固相萃取-超高效液相色谱-荧光检测法。主要重点在于样品处理和储存过程中二氢形式的稳定性。作为稳定剂，测试了不同浓度的二硫苏糖醇以及工作溶液在不同pH值（3.8 - 9.8）下的情况。在BEH酰胺柱上采用亲水作用色谱等度条件进行色谱分离。该方法在校准标准溶液中，二氢形式在10 - 10000 ng/ml范围内、氧化形式在0.5 - 1000 ng/ml范围内呈线性，在实际样品中，二氢形式在25 - 1000 ng/ml范围内、氧化形式在1 - 100 ng/ml范围内呈线性。基于不同类型的吸附剂（基于混合模式阳离子交换、多孔石墨化碳以及包含亲水和疏水成分的聚合物）开发了一种新型固相萃取样品制备方法。最终在MCAX固相萃取柱上进行了验证。方法准确度在76.9%至121.9%之间。日内和日间精密度均不超过10.7%。该方法在尿液常规临床检测中具有高灵敏度（氧化形式的定量下限为1 ng/ml，二氢形式为25 ng/ml）。健康人尿液中生物蝶呤、新蝶呤和二氢生物蝶呤的平均浓度与肌酐摩尔数相关（分别为66.8、142.3和257.3 μmol/mol肌酐），这与文献数据相符。使用我们的方法测得的二氢新蝶呤浓度为98.8 μmol/mol肌酐。
