全血硫胺素及其衍生物的高效液相色谱检测方法的建立

检验医学 ›› 2012, Vol. 27 ›› Issue (5): 331-335.

• 生物化学检验论著 • 下一篇

全血硫胺素及其衍生物的高效液相色谱检测方法的建立

费国强

1.复旦大学附属中山医院，上海 200032；2.中科院上海有机化学研究所，上海 200032

收稿日期:2012-03-12 修回日期:2012-04-15 出版日期:2012-05-30 发布日期:2012-05-10
作者简介:费国强，男，1974年生，硕士，主要研究方向为硫胺素缺乏与神经变性疾病。
基金资助:
上海市科委基础重大攻关基金资助项目(07DJ14005)

HPLC determination of thiamine and its derivatives in human whole blood

1.Zhongshan Hospital, Fudan University, Shanghai 200032, China; 2. Shanghai Institute of Organic Chemistry, Chinese Academy of Science, Shanghai 200032, China

Received:2012-03-12 Revised:2012-04-15 Online:2012-05-30 Published:2012-05-10

摘要/Abstract

摘要： 目的　建立一种稳定、快捷的全血硫胺素及其磷酸酯衍生物高效液相色谱(HPLC)荧光检测方法。方法　全血样本经三氯乙酸(TCA)去蛋白处理、铁氰化钾衍生，产生能被HPLC反向柱子经梯度洗脱分离的稳定荧光产物，通过荧光检测并与硫胺素及其磷酸酯衍生物标准样本比对，来确定人体全血硫胺素含量。方法通过线性方程、提取率、重复性等指标验证。结果　经过检测，焦磷酸硫胺素(TDP)、单磷酸硫胺素(TMP)和硫胺素的检测限分别为8.0、1.5和2.5 nmol/L，信噪比（S/N）>10，能够满足临床需要。线性趋势良好，r>0.99。硫胺素、TMP及TDP的回收率分别为103.7%、96.8%、101.7%，重复性好，变异系数（CV）<10%。结论　经TCA去蛋白、铁氰化钾衍生、梯度洗脱建立的荧光HPLC检测硫胺素及其磷酸酯衍生物衍生物的方法可靠，可用于临床检测。

关键词: 硫胺素, 铁氰化钾, 高效液相色谱

Abstract: Objective　To establish a stable and rapid method for determining the levels of human whole blood thiamine and its phosphate ester derivatives by high performance liquid chromatography (HPLC).　Methods　After blood proteins were precipitated by trichloroacetic acid (TCA), thiamine and its phosphate esters were derivatized using potassium ferricyanide into thiochromes. The derivatives of thiamine and it phosphate esters were separated by a reversedphase HPLC column and measured by fluoroscope. The method was validated by the analysis of linearity, extraction ratio and repeatability. Results　The lower limits of quantification of blood thiamine diphosphate (TDP), thiamin monophosphate (TMP) and thiamine determinations were 8.0, 1.5 and 2.5nmol/L respectively. The signal to noise ratio (S/N) for determining thiamine and its derivatives was>10. The linear relationship was stable (r>0.99). The recovery rates of TDP, TMP and thiamine were 103.7%, 96.8% and 101.7% respectively. The repeatabilities were good. All the coefficients of variation (CV) were <10%. Conclusions　The HPLC with thiamine derivatization by potassium ferricyanide for determining thiamine and its phosphate ester derivatives is reliable and can be used for clinical analysis.

Key words: Thiamine, Potassium ferricyanide, High performance liquid chromatography

费国强;李晓鸽;钟春玖. 全血硫胺素及其衍生物的高效液相色谱检测方法的建立[J]. 检验医学, 2012, 27(5): 331-335.

FEI Guo-Qiang;LI Xiao-Ge;ZHONG Chun-Jiu. HPLC determination of thiamine and its derivatives in human whole blood[J]. , 2012, 27(5): 331-335.

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全血硫胺素及其衍生物的高效液相色谱检测方法的建立

HPLC determination of thiamine and its derivatives in human whole blood

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