固相萃取-分光光度法测定人体尿液中亚硝酸盐的含量

亚硝酸盐中毒发生时，为了快速准确测定人体尿液中的亚硝酸盐的含量，建立了固相萃取-分光光度的分析方法。本方法是基于亚硝酸盐的重氮偶合反应，采用固相萃取技术对尿液进行净化处理后，消除了基质干扰，提高了测试灵敏度，通过对比考察了固相萃取柱类型、重氮化剂种类、重氮化剂质量浓度、反应酸度、偶合剂质量浓度及反应时间等试验参数对测试结果的影响，得出了最佳的试验条件，确定了试验方法为尿液离心后经HLB固相萃取柱净化除杂，取过柱液2.0 mL于比色管中，用水定容至5.0 mL，依次加入0.5 mL对氨基苯磺酰胺酸性溶液（50.0 g/L）和0. 5 mL盐酸萘乙二胺溶液( 2.5 g /L)进行重氮偶合显色反应10 min，于波长544 nm 处进行吸光度的测定，以工作曲线外标法定量。 结果显示，亚硝酸盐在0.033-0.986 μg/mL范围内线性关系良好，相关系数为0.9998，尿液在0.246-2.054 μg/mL三种加标水平范围内亚硝酸盐的平均回收率为96.3%-106.1%，表明HLB固相萃取柱有效去除了影响重氮偶合反应的干扰物质，达到了满意的净化效果。日内和日间测定值的相对标准偏差（RSD）分别为2.01% -3.58%和2.21%- 4.75%，方法检出限为0.010 μg/mL，定量下限为0.033 μg/mL。该方法操作简单、快速，精密度和准确度良好，且检出限低，适用于人体尿液中亚硝酸盐含量的快速检测。

Determination of nitrite content in human urine by spectrophotometry with solid-phase extraction

In order to achieve rapid and accurate determination of nitrite content in human urine when nitrite poisoning occurs, a method of solid-phase extraction (SPE) coupled with spectrophotometry was established. The method was based on the diazo-coupling reaction of nitrite , and purified urine using SPE technique to eliminate the interference of matrix and improve the testing sensitivity. The influence of different operating parameters such as type of SPE columns, type of diazotization agents, concentration of diazotization agent, reaction acidity, concentration of coupling agent, reaction time were compared and studied, and the optimum experimental conditions were obtained, and the testing method was determined. The urine sample was centrifuged, and the supernatant was purified with the HLB SPE column. The extract urine 2.0 mL was transferred to a colorimetric tube, and diluted to the volume of 5.0 mL with water, and 0.5 mL of the sulfanilamide acid solution(50.0 g/L) and 0.5 mL of N-(1-Naphthyl)ethylenediamine dihydrochloride solution(2.5 g/L) were added in, the mixture was mingled completely and settled for 10 min for diazo-coupling color reaction, and then measured at 544 nm by spectrophotometry and quantified by working curve in external standard method. The results showed that nitrite had a good linear relationship in the range of 0.033-0.986 μg/mL and the correlation coefficient was 0.9998. The spiked recoveries of nitrite in urine samples were ranged from 96.3% to 106.1% at three spiked levels from 0.246-2.054 μg/mL, indicating that the interfering substances that affect the diazo-coupling reaction were successfully removed by the HLB SPE column, and the purification effect was satisfied. The relative standard deviations (RSD) for intra-day and inter-day were ranged from 2.01% to 3.58% and from 2.21% to 4.75%, respectively, and the limit of detection was 0.010 μg/mL and the limit of quantification was 0.033 μg/mL. The method has the advantages of simple operation, fast detection speed, good precision, high accuracy and low detection limit, it can be used for the rapid determination of nitrite in human urine.