高原鼠兔血清中N-isopropyl oxamate的RP-HPLC检测方法的建立

Establishment of RP-HPLC detection method of N-isopropyl oxamate in the serum of plateau pikas

目的：探索N-isopropyl oxamate和血清蛋白结合水平,建立高原鼠兔血液中精子型乳酸脱氢酶（LDH-C4）特异性抑制剂N-isopropyl oxamate的高效液相色谱（HPLC）检测方法。方法：取体重150-200 g高原鼠兔20只,随机分为对照组和抑制剂组（n=10）。通过外源性加入不同浓度的N-isopropyl oxamate,检测其与血清蛋白的结合水平。采用将血清先用胰蛋白酶酶解,再加入三氯乙酸的前处理法进行HPLC检测。结果：当空白高原鼠兔血清加入抑制剂标准品达到血清中浓度为0.05 mmol/L、0.1 mmol/L、1 mmol/L、10 mmol/L、16.7 mmol/L、33.3 mmol/L、100 mmol/L时,抑制剂与血清蛋白的结合率分别为100%、100%、100%、86.84%、54.11%、40.10%、20.18%。在本文建立的方法下,回收率、精密度、稳定性均良好。N-isopropyl oxamate在0.0125-0.25 mmol/L内浓度与峰面积线性关系好。空白血清加入标准品达到终浓度为0.15 mmol/L、0.3 mmol/L和1 mmol/L时,平均回收率分别为98.05%、98.98%、98.12%;精密度相对标准偏差（RSD）分别为1.17%、0.92%、0.83%;稳定性RSD分别为1.38%、1.40%、0.88%。方法的重复性RSD为1.76%,最低定量限为0.0125 mmol/L。结论：N-isopropyl oxamate与高原鼠兔血清具有很强的亲和力,直接用三氯乙酸沉淀蛋白的前处理方法无法准确检测其浓度,而先用胰蛋白酶消化血清,再用三氯乙酸沉淀蛋白的方法可实现血液中HPLC的精确检测。

Objective： To explore the intergrating of N-isopropyl oxamate and serum protein and establish a high performance liquid chromatography（HPLC） detection method of N-isopropyl oxamate （specific inhibitor of testis-specific lactate dehydrogenase （LDH-CA）） in the blood of plateau pikas. Methods： Twenty highland pika 150 - 200 g,were randomly divided into two groups（ n = 10） ： control group and inhibitor group. Different concentrations of N-isoprepyl oxamate were added to examine the intergrating of N-isopropyl oxamate and serum protein. In order to detennine its concentration in the pika blood accurately, we used the method of adding trypsin to incubate the serum first, followed by trichloreacetic acid treatment and detecting by HPLC. Results： When the concentrations of N-isoprepyl oxamate in the pika serum were added to 0.05 mmol/L, 0.1 mmol/L, 1 mmol/L, 10 mmol/L, 16.7 mmol/L, 33.3 mmol/L and 100 mmol/L, the intergrating rates between N- isopropyl oxamate and plateau pika serum were 100%, 100%, 100%, 86.84%, 54.11%, 40.10% and 20.18%, respectively. The method established in this paper was good on reeovery rates, precision and stability. A good linearity was obtained in the range of 0. 0125- 0.25 mmol/L. When the concentrations of N-isopropyl oxamate in the serum were added to 0.15 mmol/L,0.3 mmol/L and 1 mmol/L, the recovery rates were 98.05%, 98.98% and 98.12%, respectively; the precision relative standard deviation（RSD） of concentrations were 1. 17%, 0.92% and 0.83 %, respectively; the stability relative standard deviation （RSD） of concentrations were 1.38%, 1.40% and 0. 88%, respectively. The repeatability RSD of the method was 1.76%. Quantitative limit was 0.0125 mmol/L. Conclusion： N-isopropyl oxamate has a strong affinity with plateau pika serum protein that can＇ t be accurately determined with common HPLC method. It can be accurately determined in the blood by adding trypsinto digest the serum protein first, followed by adding triehloroacetic acid to precipitate the protein.