液相微萃取-后萃取光化学荧光高效液相色谱法测定生物样品中甲氨蝶呤的含量

Determination of methotrexate in biological matrices by liquid-phase microextraction combined with photochemical fluorescence high performance liquid chromatography

目的：采用液相微萃取-后萃取光化学荧光高效液相色谱法测定生物样品中甲氨蝶呤的含量。方法：应用自制的液相微萃取装置，在0．05mol·L^-1盐酸酸性介质中，甲氨蝶呤以分子状态首先被聚醚砜中空纤维孔壁中的正丁醇萃取，继而被25μL 0.05mol·L^-1氨水溶液后萃取。后萃取液用等体积的0．2mol·L^-1盐酸酸化后，经紫外光照射45min，发生光化学反应，取样20μL进行高效液相色谱分析，在λex=275nm，λem=370nm荧光检测，建立了液相微萃取-后萃取-光化学荧光高效液相色谱法测定生物样品中甲氨蝶呤含量的方法。结果：该方法在血浆和尿液中的浓缩倍数可达20-24倍，线性范围分别为0.05-50mg·L^-1和0．01-50mg·L^-1，检出限分别为10μg·L^-1和5μg·L^-1，RSD〈11％。通过液相微萃取-后萃取，能有效地去除生物样品中干扰甲氨蝶呤测定的内源性杂质，提高了选择性。结论：该方法将液相微萃取和光化学荧光-高效液相色谱法相结合，为生物样品中甲氨蝶呤的检测提供了一种有效的分离分析方法。

OBJECTIVE To develop a method for determining the concentration of methotrexate in biological matrices by liq uid-phase microextraction combined with photochemical fluorescence high performance liquid chromatography （LPME-POF- HPLC）. METHODS With self-made liquid phase micro-extraction apparatus, methotrexate was extracted from biological sam ple in the medium of 0.05 mol·L^-1 HCI. The unionized analytes were extracted into n-butanol impregnated in the pores of the hollow fibre,and into an alkalizing solution inside the hollow fibre. The extracts were diluted with the equal volume 0.2 mol·L^-1 hydrochloric acid,after irradiation below the ultraviolet light for 45 minutes,then were injected into the HPLC instrument and determined with fluorescence detector. RESULTS Efficient sample clean-up was seen for methotrexate. The enrich factor was 20-24, the linear range and the detection limit （S/N = 3） were 0.05-50 mg·L^-1 and 10μg·L^-1 in plasma ,and 0.01-50 mg·L^-1 and 5μg·L^-1 in urine for this method,respectively,the relative standard deviation was below 11%. With the method, some endogenous substances in matrices which interfered the determination of methotrexate were eliminated efficiently, and the higher selectivity was obtained. CONCLUSION The method is validated and successfully applied to determine methotrexate in plasma and urine from patient treated with methotrexate,indicating that LPME-PCF HPLC is a promising combination for analysis of drugs present in low concentrations in biological matrices.