Nath Christa E, Dallapozza Luciano, Eslick Adam E, Misra Ashish, Carr Deborah, Earl John W
Department of Biochemistry, The Children's Hospital at Westmead, Westmead, NSW, Australia.
Biomed Chromatogr. 2009 Feb;23(2):152-9. doi: 10.1002/bmc.1096.
A novel assay for the determination of l-asparaginase activity in human plasma is described that is based on the HPLC quantitation of l-aspartic acid produced during enzyme incubation. Methods for monitoring l-asparagine depletion are also described. Chromatography of l-aspartic acid, l-asparagine and l-homoserine (the internal standard) involved derivatization with o-pthaldialdehyde, then separation from other amino acids on a Phenomenex Luna C(18) column using a 1 mL/min flow rate and a mobile phase consisting of di-potassium hydrogen orthophosphate propionate buffer, pH 6, with 10% methanol and 10% acetonitrile. Fluoresence detection was at excitation/emission wavelengths of 357/455 nm. Under these conditions l-aspartic acid, l-asparagine and l-homoserine had retention times of 3.5, 9.8 and 17.7 min, respectively. The l-asparaginase assay was linear from 0.1 to 10 U/mL activity and interday precision and accuracy were less than 13%. The limit of quantitation was approximately 0.03 U/mL. The assay utility was established in 12 children who received E. coli l-asparaginase as treatment for acute lymphoblastic leukaemia.
描述了一种基于高效液相色谱法（HPLC）定量测定酶孵育过程中产生的L-天冬氨酸来测定人血浆中L-天冬酰胺酶活性的新方法。还描述了监测L-天冬酰胺消耗的方法。L-天冬氨酸、L-天冬酰胺和L-高丝氨酸（内标）的色谱分析包括用邻苯二甲醛衍生化，然后在Phenomenex Luna C(18)柱上以1 mL/分钟的流速和由pH 6的磷酸氢二钾丙酸盐缓冲液、10%甲醇和10%乙腈组成的流动相从其他氨基酸中分离。荧光检测的激发/发射波长为357/455 nm。在这些条件下，L-天冬氨酸、L-天冬酰胺和L-高丝氨酸的保留时间分别为3.5、9.8和17.7分钟。L-天冬酰胺酶测定在0.1至10 U/mL活性范围内呈线性，日间精密度和准确度小于13%。定量限约为0.03 U/mL。该测定方法在12名接受大肠杆菌L-天冬酰胺酶治疗急性淋巴细胞白血病的儿童中得到了验证。
