BACKGROUND AND AIMS The active coenzymes of the water soluble vitamins B1 and B6 (thiamine pyrophosphate (TPP) and pyridoxal-5-phosphate (P5P) respectively) play an important role in numerous bodily functions. The simultaneous analysis of both these analytes is limited to either mass spectrometry based methods or commercial kit suppliers. In this study we developed a novel method for analysis of both TPP and P5P by fluorescence detection. METHODS Briefly, whole blood samples are precipitated by trichloroacetic acid, and P5P and TPP are both derivatised before separation on a C18-PFP column. The new assay's performance was compared against a recent cycle from an external quality assurance program (RCPAQAP) and the current only existing commercial kit (n = 76). RESULTS Linearity for both analytes was above 0.99 (r2) up to a concentration range of: 4000 nmol/L (P5P) and 2000 nmol/L (TPP). Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 6% (coefficient of variation). Recovery of both compounds exceeded 90%. Accuracy of the protocol displayed satisfactory results in proficiency testing and had an acceptable level of agreement with the existing current kit method. CONCLUSIONS Overall, this method provides an economical alternative in routine clinical diagnostic laboratories wishing to perform P5P and TPP analysis.

中文翻译：

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高效液相色谱荧光法可同时分析全血中的5-磷酸吡ido醛和焦磷酸硫胺素。

背景与目的水溶性维生素B1和B6的活性辅酶（分别为硫胺素焦磷酸（TPP）和吡ido醛5-磷酸（P5P））在许多身体功能中起重要作用。这两种分析物的同时分析仅限于基于质谱的方法或商业试剂盒供应商。在这项研究中，我们开发了一种通过荧光检测分析TPP和P5P的新方法。方法简而言之，三氯乙酸沉淀全血样品，然后在C18-PFP色谱柱上分离前先将P5P和TPP衍生化。将新测定的性能与外部质量保证计划（RCPAQAP）和当前仅现有的商用试剂盒（n = 76）的最近周期进行了比较。结果两种分析物的线性均高于0。99（r2），最高浓度范围：4000 nmol / L（P5P）和2000 nmol / L（TPP）。与商用质量控制材料相比，该方法的精度（日内和日间）低于6％（变异系数）。两种化合物的回收率均超过90％。协议的准确性在能力测试中显示出令人满意的结果，并且与现有的当前试剂盒方法具有可接受的协议水平。结论总体而言，该方法为希望进行P5P和TPP分析的常规临床诊断实验室提供了一种经济的选择。协议的准确性在能力测试中显示出令人满意的结果，并且与现有的当前试剂盒方法具有可接受的协议水平。结论总体而言，该方法为希望进行P5P和TPP分析的常规临床诊断实验室提供了一种经济的选择。协议的准确性在能力测试中显示出令人满意的结果，并且与现有的当前试剂盒方法具有可接受的协议水平。结论总体而言，该方法为希望进行P5P和TPP分析的常规临床诊断实验室提供了一种经济的选择。